Combinations of irs/stat3 dual modulators and anti-cancer agents for treating cancer

ABSTRACT

Provided is a treatment of cancer using a dual modulator of Insulin Receptor Substrate (IRS) and signal transducer and activator of transcription 3 (Stat3), as well with other agents. The combination can be used to treat a tumor that has developed resistance to an EGFR inhibitor, EGFR antibody, mTOR inhibitor, MEK inhibitor, mutated B-Raf inhibitor, chemotherapeutic agents, and certain combinations thereof, or to prevent acquired resistance of a tumor to any of said inhibitors or agents, or to prevent tumor recurrence following cease of treatment with any of said inhibitors or agents or a combination thereof. The subject matter further provides a treatment of cancer using combination therapy comprising a dual modulator of IRS and Stat3, in combination with an immunotherapy agent. The combination can be used to sensitize a tumor to immunotherapy.

CROSS REFERENCE TO RELATED APPLICATION(S)

This application is a Continuation of U.S. application Ser. No.15/548,915 filed on Aug. 4, 2017, which a 35 U.S.C. 371 National PhaseEntry Application from PCT/IL2016/050134, filed Feb. 4, 2016, whichclaims priority to U.S. Provisional Application No. 62/136,530, filedMar. 22, 2015 and U.S. Provisional Application No. 62/112,257, filedFeb. 5, 2015. The entirety of the disclosure of the above-referencedapplications are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to the treatment of cancer usingcombination therapy comprising a dual modulator of Insulin ReceptorSubstrate (IRS) and signal transducer and activator of transcription 3(Stat3), in combination with (i) a modulator of a protein kinase (PK)selected from an Epidermal Growth Factor inhibitor (EGFR inhibitor) andEGFR antibody; (ii) an inhibitor of mammalian target of rapamycin(mTOR); (iii) a mitogen-activated protein kinase (MEK) inhibitor; (iv) amutated B-Raf inhibitor; (v) a chemotherapeutic agent like Gemcitabine,5-FU, Irinotecan and Oxaliplatin; and (vi) certain combinations thereof.The combination can be used to treat a tumor that has developedresistance to an EGFR inhibitor, EGFR antibody, mTOR inhibitor, MEKinhibitor, mutated B-Raf inhibitor, chemotherapeutic agents, and certaincombinations thereof, or to prevent acquired resistance of a tumor toany of said inhibitors or agents, or to prevent tumor recurrencefollowing cease of treatment with any of said inhibitors or agents or acombination thereof. The combination provides a therapeutic effect whichis at least additive, and is preferably synergistic. The presentinvention further relates to the treatment of cancer using combinationtherapy comprising a dual modulator of IRS and Stat3, in combinationwith an immunotherapy agent. The combination can be used to sensitize atumor to immunotherapy.

BACKGROUND OF THE INVENTION

Tyrphostins are a family of protein tyrosine kinase inhibitors, designedto mimic the tyrosine substrate, the ATP and can inhibit allostericallythe enzyme (Levitzki et al., Science (1995), 267:1782-88; Levitzki etal., Biochem. Pharm. (1990), 40:913-920; Levitzki et al., FASEB J.(1992), 6:3275-3282; U.S. Pat. Nos. 5,217,999 and 5,773,476, Posner etal., Mol. Pharmacol. (1994), 45:673-683). The pharmacophores of thesetyrphostins, and in particular tyrphostins of the benzylidene malonitriltype, are the hydrophilic catechol ring and the more lipophilicsubstituted cyano-vinyl radical. Kinetic studies have shown that sometyrphostin compounds are pure competitive inhibitors vis-à-vis tyrosinesubstrates whereas for the ATP binding site they act as non-competitiveinhibitors (Yaish et al., Science (1988), 242:933-935; Gazit et al., J.Med. Chem. (1989), 32:2344-2352). Nonetheless, many tyrphostins haveshown competitive inhibition against both the substrate and ATP bindingsite or mixed competitive (Posner et al., Mol. Pharmacol. (1994),45:673-683).

In a related group of tyrphostins, the hydrophilic catechol ring wasexchanged by lipophilic dichloro- or dimethoxy-phenyl groups, to yieldEGFR kinase inhibitors, effective in the low micromolar range (Yoneda etal., Cancer Res. (1991), 51: 4430-4435). These tyrphostins were furtheradministered to tumor-bearing nude mice together with anti-EGFRmonoclonal antibodies at a suboptimal dose to afford markedly enhancedinhibition of tumor growth.

WO 2008/068751 to some of the inventors of the present invention,discloses compounds having increased inhibitory properties ofinsulin-like growth factor 1 receptor (IGF1R), platelet derived growthfactor receptor (PDGFR), epidermal growth factor receptor (EGFR), andIGF1R-related insulin receptor (IR) activation and signaling.

WO 2009/147682 to some of the inventors of the present inventiondiscloses compounds acting as protein kinase (PK) and receptor kinase(RK) signaling modulators. Further disclosed in WO 2009/147682 aremethods of preparation of the such compounds, pharmaceuticalcompositions including such compounds, and methods of using thesecompounds and compositions, especially as chemotherapeutic agents forpreventions and treatments of PK and RK related disorders such asmetabolic, inflammatory, fibrotic, and cell proliferative disorders, inparticular cancer.

WO 2012/117396 to some of the inventors of the present inventiondescribes combinations of the compounds of WO 2008/068751 or WO2009/147682 with anti-cancer agents for the treatment of cancer.

Cancers treated with conventional radio- or chemo-therapy or otheranti-cancer agents frequently develop resistance to these treatments,ultimately leading to recurrent disease that often has a more aggressivephenotype than that observed at the time of the original diagnosis (Liet al., J. Med. Chem. (2009), 52(16): 4981-5004).

In accordance with principles for selecting agents for use incombination chemotherapy regimens, drugs with different mechanisms ofaction and with additive or synergistic cytotoxic effects on the tumorcan be combined (Pazdur et al., Chapter 3: Principles of OncologicPharmacotherapy (2005), 9^(th) Edition:23-42). Multi-agent therapy hasthree important theoretical advantages over single-agent therapy. First,it can maximize cell death while minimizing host toxicities by usingagents with non-overlapping dose-limiting toxicities. Second, it mayincrease the range of drug activity against tumor cells with endogenousresistance to specific types of therapy. Finally, it may also prevent orslow the development of newly resistant tumor cells. Virtually, almostall curative chemotherapy regimens for cancer employ multi-agent drugcombinations (Frei and Eder, Cancer medicine (2003), 11:817-837).

A family of relatively new anti-cancer agents are inhibitors (e.g.antibodies and small molecules) of specific kinases or other signalingenzymes involved in the mitogenic, anti-apoptotic, angiogenic ormetastatic pathways in the cancerous cells. Examples of approved drugsincluded in this family are the EGFR and/or HER2 blockers (e.g. thesmall molecules gefitinib, erlotinib, lapatinib or antibodies liketrastuzumab (Herceptin®) and cetuximab (Erbitux®)), B-Raf inhibitors(e.g. PLX-4032, sorafenib), BCR-ABL and/or Src family kinase inhibitors(e.g. imatinib, dasatinib, nilotinib), VEGFR/PDGFR and/or multi kinaseinhibitors (e.g. bevacizumab (Avastin®), sorafenib, sunitinib, andpazopanib), and proteasome inhibitors (e.g. bortezomib (Velcade®)) etc.Several EGFR inhibitors were approved by the FDA like Tarceva(Erlotinib) in 2004, Iressa (Gefitinib) in 2003, and Lapatinib in 2010,as well as antibodies against EGFR.

Another family of anti-cancer agents are inhibitors of the mammaliantarget of rapamycin (mTOR). mTOR (also called FRAP (FKBP-rapamycinassociated protein), RAFT (rapamycin and FKBP target), RAPT1, or SEP) isa serine/threonine kinase, which belongs to phosphatidylinositol-3kinase (PI3K) related kinases (PIKKs) family mTOR functions as a centralcontroller of growth, proliferation, metabolism and angiogenesis, butits signaling is deregulated in various human diseases especiallycertain cancers like renal cell carcinoma and breast cancer. In cancer,mTOR is frequently hyperactivated which promotes cancer development andprogression. Recent development has made cancer treatment move on fromconventional cytotoxic drugs to agents that target specific proteinslike mTOR called mTOR inhibitors. A common mTOR inhibitor, rapamycin(Sirolimus), is a bacterial product that inhibits mTOR by associatingwith its intracellular receptor. Two mTOR inhibitors, Temsirolimus(CCI-779) and Everolimus (Afinitor, RAD-001) which are derivatives ofrapamycin, are approved for the treatment of patients with advancedrenal cell carcinoma (RCC) and mantle cell lymphoma. Other examples ofmTOR inhibitors include Ridaforolimus (Deforolimus, AP23573), andNVP-BEZ235 which is a dual inhibitor of PI3K and mTOR.

The first generation of mTOR inhibitors like rapamycin, show certainlimitations by blocking only C1 isoform, inducing feedback activation ofAKT and showing resistance to the second isoform mTORC2. A panel ofsecond generation agents that can inhibit both mTORC1 and mTORC2 bytargeting kinase domains with high degree of selectivity are beingdeveloped. Examples of second generation mTOR inhibitors include OSI-027(OSI Pharmaceuticals), XL765 (Exelixis), INK128, MLN0128, AZD2014,DS-3078a and Palomid529.

In the last few decades immunotherapy has become an important part oftreating some types of cancer. The goal of cancer immunotherapy is toenable the patient's immune system to specifically recognize and killcancer cells. Signal transducer and activator of transcription 3 (Stat3)is often activated in cancer and directly involved in the implementationand maintenance of the cancer immunosuppressive microenvironment andplays a central role in tumor immune evasion.

There is an unmet need for combinations that are useful for treatingcancer, preferably providing at least additive therapeutic effects.Combinations of drugs from different categories are useful to prevent orovercome emergence of drug resistant tumors.

SUMMARY OF THE INVENTION

The present invention relates to compositions and methods for treatingcancer, by administering a combination comprising at least one compoundwhich is a dual modulator of Insulin Receptor Substrate (IRS) and signaltransducer and activator of transcription 3 (Stat3), e.g., a compound offormula (III) or (IV), or any of the compounds covered by theseformulae, in combination with (i) a modulator of a protein kinase (PK)selected from an Epidermal Growth Factor inhibitor (EGFR inhibitor) andEGFR antibody; (ii) an inhibitor of mammalian target of rapamycin(mTOR); (iii) a mitogen-activated protein kinase (MEK) inhibitor; (iv) amutated B-Raf inhibitor; (v) a chemotherapeutic agent like Gemcitabine,5-FU, Irinotecan and Oxaliplatin; and (vi) certain combinations thereof.The present invention further relates to the treatment of cancer usingcombination therapy comprising a dual modulator of IRS and Stat3, e.g.,a compound of formula (III) or (IV), or any of the compounds covered bythese formulae, in combination with an immunotherapy agent.

The compounds described herein are modulators of Insulin ReceptorSubstrate 1 (IRS1) and/or Insulin Receptor Substrate 2 (IRS2) signaling.Accordingly, these compounds are referred to herein as “modulators ofIRS”. In some embodiments, the compounds are inhibitors of IRS1 and/orIRS2. In further embodiments, the compounds of the invention areinhibitors of insulin-like growth factor 1 receptor (IGF-1R). As such,these compounds are useful in inhibiting, treating or preventing IGF-1Rand/or IRS1 and/or IRS2 signaling related disorders, for example cancer.In some embodiments, the compounds trigger any one or more of thefollowing, in any order: (i) dissociation of IRS1 and/or IRS2 from thecell membrane; (ii) serine phosphorylation of the IGF-1R directsubstrates IRS1 and/or IRS2; and/or (iii) degradation of IRS1 and/orIRS2, thus providing long-lasting effects which enhance the inhibitoryactivity of these compounds. In other embodiments, the compounds arealso inhibitors of IGF1R-related insulin receptor (IR), or proteinsaffected by or mediated by these PTKs or that are part of thePTK-mediated signal transduction pathway.

IRS are negatively regulated by EGFR downstream elements as well as bymTOR/S6K. Therefore, treating patients with drugs that inhibit thesetargets may result, as a side effect, in upregulating IRS and activatinga central survival pathway to AKT. According to the principles of thepresent invention, this feedback mechanism leading to drug resistancemay be overwhelmed by combining IRS destructor to the treatment, asdemonstrated herein.

The compounds described herein are also modulators of signal transducerand activator of transcription 3 (Stat3). Accordingly, these compoundsare also referred to herein as “modulators of Stat3”. In someembodiments, the compounds lead to the inhibition of Stat3phosphorylation in cancer cells. Increased levels of Stat3phosphorylation are detected in various cancers and drug-resistantcancers, leading to enhanced cancer survival. Moreover, treatment ofcancers with PK inhibitor drugs surprisingly leads to the induction ofStat3 phosphorylation, as demonstrated herein. Without wishing to bebound by any particular theory or mechanism of action, it iscontemplated that inhibiting Stat3 activity with the compounds of thepresent invention may synergize with such PK inhibitor drugs, which as aside effect upregulate Stat3, may prevent acquired resistance to suchdrugs, and may be effective for drug-resistant cancers.

Due to their dual effect on IRS and Stat3, the compounds are furtherdescribed herein as “IRS/Stat3 dual modulators”.

Thus, in one embodiment, the present invention relates to a method oftreating a tumor that has developed resistance to an Epidermal GrowthFactor Receptor (EGFR) inhibitor and/or EGFR antibody, the methodcomprising the step of contacting the tumor with an EGFR inhibitorand/or EGFR antibody in combination with a compound represented by thestructure of formula (III) or (IV).

In another embodiment, the present invention relates to a method ofpreventing acquired resistance of a tumor to an Epidermal Growth FactorReceptor (EGFR) inhibitor and/or EGFR antibody, the method comprisingthe step of contacting the tumor with an EGFR inhibitor and/or EGFRantibody in combination with a compound represented by the structure offormula (III) or (IV).

In another embodiment, the present invention relates to a method ofpreventing or delaying tumor recurrence following cease of treatmentwith a EGFR inhibitor and/or EGFR antibody, the method comprising thestep of contacting the tumor with an EGFR inhibitor and/or EGFR antibodyin combination with a compound represented by the structure of formula(III) or (IV).

In another embodiment, the present invention relates to a pharmaceuticalcombination comprising a compound represented by the structure offormula (III) or (IV), in combination with an Epidermal Growth Factor(EGFR) inhibitor, and/or EGFR antibody.

In another embodiment, the present invention relates to pharmaceuticalcombination comprising a compound represented by the structure offormula (III), in combination with an Epidermal Growth Factor (EGFR)inhibitor and/or EGFR antibody, wherein the EGFR inhibitor is selectedfrom the group consisting of erlotinib, gefitinib, lapatinib,vandetanib, neratinib, icotinib, afatinib, dacomitinib, poziotinib,AZD9291, CO-1686, HM61713 and AP26113, and wherein the EGFR antibody isselected from the group consisting of trastuzumab, necitumumab,cetuximab and panitumumab, preferably wherein the EGFR inhibitor iserlotinib or afatinib, and/or wherein the EGFR antibody is cetuximab.

In other embodiments, the present invention relates to pharmaceuticalcombination comprising a compound represented by the structure offormula (IV), in combination with an Epidermal Growth Factor (EGFR)inhibitor and/or EGFR antibody.

In other embodiments, the present invention further relates to thepharmaceutical combinations as described above for use in treating atumor that is resistant to an EGFR inhibitor and/or EGFR antibody, orfor preventing acquired resistance to an EGFR inhibitor and/or EGFRantibody, or for preventing or delaying tumor recurrence following ceaseof treatment with a EGFR inhibitor and/or EGFR antibody.

In other embodiments, the present invention further relates to the useof the combinations described above for the preparation of a medicamentfor the treatment of a tumor that is resistant to an EGFR inhibitorand/or EGFR antibody, or for preventing acquired resistance to an EGFRinhibitor and/or EGFR antibody, or for preventing or delaying tumorrecurrence following cease of treatment with a EGFR inhibitor and/orEGFR antibody.

In one embodiment, the compound is represented by the structure offormula (III). In another embodiment, the compound is represented by thestructure of formula (IV). Each possibility represents a separateembodiment of the present invention.

In some embodiments, the tumor is present in a cancer patient havingtumors with acquired resistance to EGFR inhibitor and/or EGFR antibodytreatment. In other embodiments, the tumor is present in a cancerpatient who is receiving treatment with an EGFR inhibitor and/or EGFRantibody or is a candidate for receiving such treatment. In otherembodiments, the treatment results in attenuation or regression in thegrowth of the resistant tumors.

In some embodiments, the EGFR inhibitor is selected from the groupconsisting of erlotinib, gefitinib, lapatinib, vandetanib, neratinib,icotinib, afatinib, dacomitinib, poziotinib, AZD9291, CO-1686, HM61713and AP26113. In one currently preferred embodiment, the EGFR inhibitoris erlotinib. In another currently preferred embodiment, the EGFRinhibitor is afatinib. Each possibility represents a separate embodimentof the present invention.

In some embodiments, the EGFR antibody is selected from the groupconsisting of trastuzumab, necitumumab, cetuximab and panitumumab. Inone currently preferred embodiment, the EGFR antibody is cetuximab.

In some embodiments, the compound is a compound of formula (III),represented by the structure of formula D in combination with erlotinibor afatinib.

In other embodiments, the compound is a compound of formula (III),represented by the structure of formula D, in combination withcetuximab.

In other embodiments, the compound is a compound of formula (III),represented by the structure of formula D, in combination with afatiniband cetuximab.

In some embodiments, the combination treatment includes a compound offormula (III) or (IV), and either an EGFR antibody, or EGFR inhibitor.In other embodiments, the combination treatment includes a compound offormula (III) or (IV), and both an EGFR antibody and an EGFR inhibitor.In some currently preferred embodiments, the EGFR inhibitor is erlotinibor afatinib, and the EGFR antibody is cetuximab.

In one specific embodiment, the present invention relates to apharmaceutical combination comprising a compound represented by thestructure of formula D in combination with erlotinib. In one specificembodiment, the present invention relates to a pharmaceuticalcombination comprising a compound represented by the structure offormula D in combination with afatinib. In another specific embodiment,the present invention relates to a pharmaceutical combination comprisinga compound represented by the structure of formula D in combination withcetuximab. In another specific embodiment, the present invention relatesto a pharmaceutical combination comprising a compound represented by thestructure of formula D in combination with afatinib and cetuximab.

In other aspects, it has now unexpectedly been found that a combinationof a dual modulator of Insulin Receptor Substrate (IRS) and signaltransducer and activator of transcription 3 (Stat3), as describedherein, and an inhibitor of mammalian target of rapamycin (mTOR),provides a therapeutic effect that is at least additive, and ispreferably synergistic as compared with the treatment effect of eachagent alone. Furthermore, the combination can be used to treat a tumorthat has developed resistance to an mTOR inhibitor, and/or to preventacquired resistance of a tumor to the mTOR inhibitor and/or to preventor delay tumor recurrence following cease of treatment with an inhibitorof mammalian target of rapamycin (mTOR).

Accordingly, in one embodiment, the present invention relates to apharmaceutical combination comprising a compound represented by thestructure of formula (III) or (IV), and at least one inhibitor ofmammalian target of rapamycin (mTOR), wherein the compound and the atleast one mTOR inhibitor together provide a synergistic therapeuticanti-cancer effect.

In other embodiments, the present invention relates to a method oftreating cancer, comprising the step of administering to the subject inneed thereof a therapeutically effective amount of a pharmaceuticalcombination comprising a compound represented by the structure offormula (III) or (IV), and at least one inhibitor of mammalian target ofrapamycin (mTOR), wherein the compound and the at least one mTORinhibitor together provide a synergistic therapeutic effect.

In other embodiments, the present invention relates to a method oftreating a tumor that has developed resistance to an inhibitor ofmammalian target of rapamycin (mTOR), the method comprising the step ofcontacting the tumor with an mTOR inhibitor in combination with acompound represented by the structure of formula (III) or (IV).

In other embodiments, the present invention relates to a method ofpreventing acquired resistance of a tumor to an inhibitor of mammaliantarget of rapamycin (mTOR), the method comprising the step of contactingthe tumor with an mTOR inhibitor in combination with a compoundrepresented by the structure of formula (III) or (IV).

In other embodiments, the present invention relates to a method ofpreventing or delaying tumor recurrence following cease of treatmentwith an inhibitor of mammalian target of rapamycin (mTOR), the methodcomprising the step of contacting the tumor with an mTOR inhibitor incombination with a compound represented by the structure of formula(III) or (IV).

In other embodiments, the present invention further relates to thepharmaceutical combination as described above for use in treating atumor that is resistant to an mTOR inhibitor, or for preventing acquiredresistance to an mTOR inhibitor, or for preventing or delaying tumorrecurrence following cease of treatment with an inhibitor of mTOR.

In other embodiments, the present invention further relates to the useof the combination described above for the preparation of a medicamentfor the treatment of a tumor that is resistant to an mTOR inhibitor, orfor preventing acquired resistance to an mTOR inhibitor, or forpreventing or delaying tumor recurrence following cease of treatmentwith an inhibitor of mTOR.

In one embodiment, the compound is represented by the structure offormula (III). In another embodiment, the compound is represented by thestructure of formula (IV). Each possibility represents a separateembodiment of the present invention.

In some embodiment, the tumor is present in a cancer patient havingtumors with acquired resistance to mTOR inhibitor treatment. In otherembodiments, the tumor is present in a cancer patient who is receivingtreatment with an mTOR inhibitor or is a candidate for receiving suchtreatment. In other embodiments, the treatment results in attenuation orregression in the growth of the resistant tumors.

Any mTOR inhibitor known to a person of skill in the art may be used inthe combinations of the present invention. In some embodiments, the mTORinhibitor is selected from the group consisting of rapamycin(Sirolimus), Ridaforolimus (Deforolimus, AP23573), NVP-BEZ235,Everolimus (Afinitor, RAD-001), Temsirolimus (CCI-779), OSI-027, XL765,INK128, MLN0128, AZD2014, DS-3078a and Palomid529. In a currentlypreferred embodiment, the mTOR inhibitor is Everolimus.

In one specific embodiment, the compound is represented by the structureof formula D and the mTOR inhibitor is Everolimus (Afinitor).

In some embodiments, the subject or cancer patient is a human.

In other aspects, it has unexpectedly been found that dual modulators ofIRS and Stat3 can be used to sensitize a tumor to immunotherapy. It isknown that Stat3 is often activated in cancer and is directly involvedin the implementation and maintenance of the cancer immunosuppressivemicroenvironment and plays a central role in tumor immune evasion.Without wishing to be bound by any particular theory or mechanism ofaction, it is contemplated that inhibition of Stat3 phosphorylation withthe compounds of the present invention un-masks the tumor from the localimmune system and sensitizes them to immunotherapy e.g. antibodiesagainst PDLs, PD1, CTLA4 or any other immunotherapy agents.

Thus, in one embodiment, the present invention relates to a method ofsensitizing a tumor to immunotherapy, the method comprising the step ofcontacting the tumor with a compound represented by the structure offormula (III) or (IV) in combination with an immunotherapy agent.

In another embodiment, the present invention further relates to apharmaceutical combination comprising a compound represented by thestructure of formula (III) or (IV), in combination with an immunotherapyagent.

In other embodiments, the present invention further relates to acombination comprising a compound of formula (III) or (IV) with animmunotherapy agent, for use in sensitizing a tumor to immunotherapy.

In other embodiments, the present invention further relates to the useof a combination comprising a compound of formula (III) or (IV) with animmunotherapy agent, for the preparation of a medicament for thetreatment of a tumor by sensitizing the tumor to immunotherapy.

In one embodiment, the compound is represented by the structure offormula (III). In another embodiment, the compound is represented by thestructure of formula (IV). Each possibility represents a separateembodiment of the present invention.

In some embodiments, the immunotherapy agent used in combination withthe compound described above is an antibody against a target selectedfrom the group consisting of PDL, PD1, CTLA4, CD20, CD30, CD33, CD52,VEGF, CD30, EGFR and ErbB2. In some embodiments, the antibody isselected from the group consisting of Alemtuzumab, Bevacizumab,Brentuximab vedotin, Cetuximab, Gemtuzumab ozogamicin, Ibritumomabtiuxetan, Ipilimumab, Ofatumumab, Panitumumab, Rituximab, Tositumomaband Tratuzumab. Each possibility represents a separate embodiment of thepresent invention.

In some embodiments, the tumor is present in a cancer patient who isreceiving immunotherapy or a candidate for receiving immunotherapy.

In other aspects, it has now unexpectedly been found that a combinationof a dual modulator of Insulin Receptor Substrate (IRS) and signaltransducer and activator of transcription 3 (Stat3), as describedherein, and a mitogen-activated protein kinase (MEK) inhibitor and/or amutated B-Raf inhibitor, provides a therapeutic effect that is at leastadditive, and is preferably synergistic as compared with the treatmenteffect of each agent alone. Furthermore, the combination can be used totreat a tumor that has developed resistance to a MEK inhibitor and/ormutated B-Raf inhibitor, and/or to prevent acquired resistance of atumor to a MEK inhibitor and/or mutated B-Raf inhibitor and/or toprevent or delay tumor recurrence following cease of treatment with aMEK inhibitor and/or mutated B-Raf inhibitor.

Thus, in some embodiments, the present invention relates to a method oftreating a tumor that has developed resistance to a mitogen-activatedprotein kinase (MEK) inhibitor and/or a mutated B-Raf inhibitor, themethod comprising the step of contacting the tumor with a MEK inhibitorand/or mutated B-Raf inhibitor, in combination with a compoundrepresented by the structure of formula (III) or (IV).

In other embodiments, the present invention relates to method ofpreventing acquired resistance of a tumor to a MEK inhibitor and/ormutated B-Raf inhibitor, the method comprising the step of contactingthe tumor with a MEK inhibitor and/or mutated B-Raf inhibitor, incombination with a compound represented by the structure of formula(III) or (IV).

In other embodiments, the present invention relates to a method ofpreventing or delaying tumor recurrence following cease of treatmentwith a MEK inhibitor and/or a mutated B-Raf inhibitor, the methodcomprising the step of contacting the tumor with a MEK inhibitor and/ormutated B-Raf inhibitor, in combination with a compound represented bythe structure of formula (III) or (IV).

In other embodiments, the present invention relates to a pharmaceuticalcombination comprising a compound represented by the structure offormula (III), in combination with a mitogen-activated protein kinase(MEK) inhibitor, and optionally a mutated B-Raf inhibitor.

In some embodiments, the combination comprises a compound of formula(III), a MEK inhibitor and a mutated B-Raf inhibitor preferably, whereinthe MEK inhibitor is Trametinib, and the mutated B-Raf inhibitor isVemurafenib.

In other embodiments, the present invention relates to a compoundrepresented by the structure of formula (IV), in combination with amitogen-activated protein kinase (MEK) inhibitor, and/or a mutated B-Rafinhibitor.

In some embodiments, the tumor is present in a cancer patient havingtumors with acquired resistance to MEK inhibitor and/or mutated B-Rafinhibitor treatment. In other embodiments, the treatment results inattenuation or regression in the growth of the resistant tumors. Inother embodiments, the tumor is present in a cancer patient who isreceiving treatment with an MEK inhibitor and/or a mutated B-Rafinhibitor or is a candidate for receiving such treatment.

Any MEK inhibitor known to a person of skill in the art may be used inthe combinations of the present invention. In some embodiments, the MEKinhibitor is selected from the group consisting of Trametinib(GSK1120212), Selumetinib, Binimetinib (MEK162), PD-325901, Cobimetinib,CI-1040 and PD035901, preferably, wherein the MEK inhibitor is Trametin.

Any mutated B-Raf inhibitor known to a person of skill in the art may beused in the combinations of the present invention. In some embodiments,the mutated B-Raf inhibitor is selected from the group consisting ofVemurafenib (PLX-4032), PLX4720, Sorafenib (BAY43-9006), and Dabrafenib,preferably, wherein the mutated B-Raf inhibitor is Vemurafenib.

In one embodiment, the compound is represented by the structure offormula (III). In another embodiment, the compound is represented by thestructure of formula (IV). Each possibility represents a separateembodiment of the present invention.

In some embodiments, the compound is represented by the structure offormula D and the MEK inhibitor is Trametinib.

In other embodiments, the compound is represented by the structure offormula D and the mutated B-Raf inhibitor is Vemurafenib.

In some embodiments, the combination treatment includes a compound offormula (III) or (IV), and either a MEK inhibitor or a mutated B-Rafinhibitor. In other embodiments, the combination treatment includes acompound of formula (III) or (IV), and both a MEK inhibitor and amutated B-Raf inhibitor.

In some embodiments, the subject or cancer patient is a human.

In some embodiments, the present invention relates to a pharmaceuticalcombination comprising a compound represented by the structure offormula D in combination with Trametinib.

In other embodiments, the present invention relates to a pharmaceuticalcombination comprising a compound represented by the structure offormula D in combination with Trametinib and Vemurafenib.

In other embodiments, the present invention relates to the combinationsdescribed above, for use in treating a tumor that is resistant to a MEKinhibitor and/or a mutated B-Raf inhibitor, or for preventing acquiredresistance to a MEK inhibitor and/or a mutated B-Raf inhibitor, or forpreventing or delaying tumor recurrence following cease of treatmentwith a MEK inhibitor and/or a mutated B-Raf inhibitor.

In other embodiments, the present invention relates to the use of thecombinations described above, for the preparation of a medicament forthe treatment of a tumor that is resistant to a MEK inhibitor and/or amutated B-Raf inhibitor, or for preventing acquired resistance to a MEKinhibitor and/or a mutated B-Raf inhibitor, or for preventing ordelaying tumor recurrence following cease of treatment with a MEKinhibitor and/or a mutated B-Raf inhibitor.

In other aspects, it has now unexpectedly been found that a combinationof a dual modulator of Insulin Receptor Substrate (IRS) and signaltransducer and activator of transcription 3 (Stat3), as describedherein, and a chemotherapeutic agent such as Gemcitabine, 5-FU,Irinotecan, Oxaliplatin and any combination thereof (e.g., thecombination treatment FOLFIRI or FOLFOX), provides a therapeutic effectthat is at least additive, and is preferably synergistic as comparedwith the treatment effect of each agent alone. Furthermore, thecombination can be used to treat a tumor that has developed resistanceto any of these chemotherapeutic agents or their combination and/or toprevent acquired resistance of a tumor to any of these chemotherapeuticagents or their combination, and/or to prevent or delay tumor recurrencefollowing cease of treatment with any of these therapeutic agents ortheir combination.

FOLFIRI is a combination treatment for cancer containing Leucovorin(Folinic Acid), 5-FU and Irinotecan. FOLFOX is a combination treatmentfor cancer containing Leucovorin calcium (Folinic Acid), 5-FU andOxaliplatin.

Thus, according to some embodiments, the present invention relates to apharmaceutical combination comprising a compound represented by thestructure of formula (III) or (IV) and at least one chemotherapeuticagent selected from Gemcitabine, 5-FU, Irinotecan, Oxaliplatin and anycombination thereof, wherein the compound and the chemotherapeuticagent(s) together provide a synergistic therapeutic anti-cancer effect.

In some embodiments, the present invention relates to a method oftreating cancer, comprising the step of administering to the subject inneed thereof a therapeutically effective amount of a combinationcomprising a compound represented by the structure of formula (III) or(IV) and at least one chemotherapeutic agent selected from Gemcitabine,5-FU, Irinotecan, Oxaliplatin and any combination thereof, wherein thecompound and the chemotherapeutic agent(s) together provide asynergistic therapeutic anti-cancer effect.

In other embodiments, the present invention provides a method oftreating a tumor that has developed resistance to at least onechemotherapeutic agent, e.g., Gemcitabine, 5-FU, Irinotecan, Oxaliplatinand any combination thereof, the method comprising the step ofcontacting the tumor with at least one of said chemotherapeutic agent(s)in combination with a compound represented by the structure of formula(III) or (IV).

In other embodiments, the present invention provides a method ofpreventing acquired resistance of a tumor to at least onechemotherapeutic agent, e.g., Gemcitabine, 5-FU, Irinotecan, Oxaliplatinand any combination thereof, the method comprising the step ofcontacting the tumor with at least one of said chemotherapeutic agent(s)in combination with a compound represented by the structure of formula(III) or (IV).

In other embodiments, the present invention provides a method ofpreventing or delaying tumor recurrence following cease of treatmentwith at least one chemotherapeutic agent, e.g., Gemcitabine, 5-FU,Irinotecan, Oxaliplatin and any combination thereof, the methodcomprising the step of contacting the tumor with at least one of saidchemotherapeutic agent(s) in combination with a compound represented bythe structure of formula (III) or (IV).

In some embodiments, the tumor is present in a cancer patient havingtumors with acquired resistance to any one or more of saidchemotherapeutic agent(s). In other embodiments, the treatment resultsin attenuation or regression in the growth of the resistant tumors. Inother embodiments, the tumor is present in a cancer patient who isreceiving treatment with said chemotherapeutic agent(s), or is acandidate for receiving such treatment.

In other embodiments, the present invention provides a pharmaceuticalcombination comprising a compound represented by the structure offormula (III) or (IV) and at least one chemotherapeutic agent selectedfrom Gemcitabine, 5-FU, Irinotecan, Oxaliplatin and any combinationthereof, for use in treating a tumor that is resistant to any one ormore of said chemotherapeutic agent(s), or for preventing acquiredresistance to said chemotherapeutic agent(s), or for delaying tumorrecurrence following cease of treatment with any one or more of suchchemotherapeutic agent(s).

In other embodiments, the present invention relates to the use of apharmaceutical combination comprising a compound represented by thestructure of formula (III) or (IV) and at least one chemotherapeuticagent selected from Gemcitabine, 5-FU, Irinotecan, Oxaliplatin and anycombination thereof, for the preparation of a medicament for thetreatment of a tumor that is resistant to said chemotherapeuticagent(s), or for preventing acquired resistance to said chemotherapeuticagent(s), or for of preventing or delaying tumor recurrence followingcease of treatment with such chemotherapeutic agent(s).

As contemplated herein, the present invention provides several exampleswhere tumors with activated K-RAS that did not respond to an anti-cancerdrug, demonstrated impressive tumor regression when the anti-canceragent was combined with compound D. For example, the combination ofanti-cancer agents with compound D converted “Non-responding” tumors to“Responder” to the anti-cancer agent. In example 1 (FIG. 1) genomicanalysis of the Erlotinib-treated group at the end point, meaning—theErlotinib-resistant clones, revealed several alterations as compared tothe control. Among these novel genomic variations was KRAS, known togenerate resistance to EGFR inhibitors. Genomic alternations related toKRAS included amplification of KRAS and NF-1 loss, which results in theactivation of K-Ras. In contrast to the Erlotinib-treated tumors, thetumors that were treated with both Erlotinib and compound D did not havethe KRAS alterations. In these tumors (treated by both Erlotinib andcompound D)− resistance to Erlotinib was not acquired and tumors did notprogress while on treatment. In addition, treatment of theErlotinib-resistant tumors with the compound D+ Erlotinib re-sensitizedthese tumors to Erlotinib and induced tumor regression. This suggeststhat compound D inclusion antagonized the resistance imposed by KRASamplification and/or activation. In another example, theGemcitabine-resistant pancreatic tumors (FIG. 13A,B), bearing mutatedKRAS, were re-sensitized to the treatment with Gemcitabine by combiningit with compound D. Furthermore, supporting data from the literatureshow that many drug-treated “oncogene-addicted” cancer cells engage apositive feedback loop leading to STATS activation, consequentlypromoting cell survival and limiting overall drug response. This wasobserved in cancer cells driven by diverse activated kinases, includingEGFR, HER2, ALK, and MET, as well as mutant KRAS.

Accordingly, in some aspects, the present invention relates to a methodof treating a tumor, comprising the step of contacting the tumor withcombinations of a compound of formula (III) or (IV) with an anti-cancerdrug, to which the tumors developed resistance due to mutations and/oramplification in KRAS. Any of the anti-cancer agents described herein(e.g., EGFR inhibitor/EGFR antibody/mTOR inhibitor/immunotherapyagent/MEK inhibitor/mutated B-Raf inhibitor/chemotherapeuticagent/combinations of the foregoing) can be used in in such methods, bytreating tumors that have developed resistance to such agents due tomutations and/or amplification of KRAS.

The compound of formula (III) is represented by the structure

wherein

-   -   R¹, R², R⁵ and R⁶ are independently selected from H, C₁-C₄        alkyl, (CH₂CH₂O)_(n)H wherein n is an integer of 1 to 20, acyl        and a functional group that gives rise to hydroxyl upon        hydrolysis;    -   R³, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³ and R¹⁴ are independently        selected from H, halogen, C₁-C₄ alkyl, C₁-C₄ haloalkyl,        CH₂SR^(a) wherein R^(a) is selected from H, C₁-C₄ alkyl, aryl,        heterocyclyl, heteroaryl, C₁-C₄ alkylaryl, C₁-C₄        alkylheterocyclyl and C₁-C₄ alkylhereroaryl, and OR¹⁶ wherein        R¹⁶ is H, C₁-C₄ alkyl, (CH₂CH₂O)_(n)H, acyl or a functional        group that gives rise to hydroxyl upon hydrolysis; and    -   R⁴ is H or CN;    -   including salts, hydrates, solvates, polymorphs, optical        isomers, geometrical isomers, enantiomers, diastereomers, and        mixtures thereof.

In some embodiments, the compound of formula (III) is selected from thegroup consisting of compounds A, B, C, D, E, F, G, H, I and J:

In one embodiment, the compound is a compound of formula A. In anotherembodiment, the compound is a compound of formula B. In anotherembodiment, the compound is a compound of formula C. In anotherembodiment, the compound is a compound of formula D. In anotherembodiment, the compound is a compound of formula E. In anotherembodiment, the compound is a compound of formula F. In anotherembodiment, the compound is a compound of formula G. In anotherembodiment, the compound is a compound of formula H. In anotherembodiment, the compound is a compound of formula I. In anotherembodiment, the compound is a compound of formula J. In one currentlypreferred embodiment, the compound is represented by the structure offormula D.

The compound of formula (IV) is represented by the structure

wherein

-   -   A is H or CN;    -   Z is S, SO or SO₂;    -   X¹, X², X³, X⁴, X⁵, Y¹ and Y² are each independently selected        from H, halogen, alkyl, haloalkyl and OR¹; and    -   Y³ and Y⁴ are each OR¹, wherein each R¹ is independently H,        C₁-C₄ alkyl, —(CH₂CH₂O)_(n)H wherein n is an integer of 1 to 20,        acyl or a functional group that gives rise to hydroxyl upon        hydrolysis, including salts, hydrates, solvates, polymorphs,        optical isomers, geometrical isomers, enantiomers,        diastereomers, and mixtures thereof.

In some embodiments, the compound of formula (IV) is represented by thestructure of formula (IV-4)

It is further apparent to a person of skill in the art that any othercompounds of formula (I) or (IV) described herein can be used for any ofthe combination treatments described by the present invention.

The combinations of the present invention are suitable for treatingvarious types of cancers. In particular, the combinations of the presentinvention are active against head and neck (H&N) cancer, sarcoma,multiple myeloma, ovarian cancer, breast cancer, kidney cancer, stomachcancer, hematopoietic cancers, lymphoma, leukemia, includinglymphoblastic leukemia, lung carcinoma, melanoma, glioblastoma,hepatocarcinoma, prostate cancer and colon cancer. Each possibilityrepresents a separate embodiment of the present invention.

The term “combination” or “combined treatment” as used herein denotesany form of concurrent or parallel treatment with at least two distincttherapeutic agents. This term is intended to encompass both concomitantadministration of the two treatment modalities, i.e., usingsubstantially the same treatment schedule, as well as overlappingadministration in sequential or alternating schedules of each treatment.Each possibility represents a separate embodiment of the presentinvention.

The combination therapy is particularly advantageous, since the dosageof each agent in a combination therapy can be reduced as compared tomono-therapy with each agent, while still achieving an overallanti-cancer effect. Accordingly, reducing the dosage of each agent mayresult in decreased side effects. The combination therapy may reduce thedevelopment of resistance to a specific anti-cancer treatment and/orlead to regression of the tumor after it has acquired resistance, asdemonstrated herein.

The compound of formula (III) or (IV) and the EGFR inhibitor/EGFRantibody/mTOR inhibitor/immunotherapy agent/MEK inhibitor/mutated B-Rafinhibitor/chemotherapeutic agent/combinations of the foregoing can beadministered simultaneously (in the same or in separate dosage forms),or they can be administered sequentially, in any order. Theadministration can also take place according to alternating dosingschedules, e.g., compound of formula (III) or (IV) followed by theadditional agent(s), then an additional dose of the compound of formula(III) or (IV), followed by the same or yet another agent(s) and soforth. All administration schedules, including simultaneous, sequentialand alternating, are contemplated by the present invention, wherein eachpossibility represents a separate embodiment of the present invention.

The pharmaceutical compositions of the present invention can be providedin any form known in the art, for example in a form suitable for oraladministration (e.g., a solution, a suspension, a syrup, an emulsion, adispersion, a tablet, a pill, a capsule, a pellet, granules and apowder), for parenteral administration (e.g., intravenous,intramuscular, intra-arterial, transdermal, subcutaneous orintra-peritoneal), for topical administration (e.g., an ointment, a gel,a cream), for administration by inhalation or for administration viasuppository. Each possibility represents a separate embodiment of thepresent invention.

Further embodiments and the full scope of applicability of the presentinvention will become apparent from the detailed description givenhereinafter. However, it should be understood that the detaileddescription and specific examples, while indicating preferredembodiments of the invention, are given by way of illustration only,since various changes and modifications within the spirit and scope ofthe invention will become apparent to those skilled in the art from thisdetailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Compound D prevents acquired resistance to Erlotinib in miceimplanted with a tumor from a Head & Neck (H&N) cancer patient. Micewere treated with (a) vehicle (⋄); (b) Erlotinib (□); (c) Compound D(Δ); or (d) Erlotinib+Compound D (∘). Treatments were initiated whenaverage tumor size was ˜80 mm³. Treatment with Erlotinib inducedsignificant tumor regression, but while on treatment allErlotinib-treated mice developed resistance to Erlotinib and theirtumors aggressively regrew. Combined treatment of Erlotinib and CompoundD induced tumor regression and none of them regrew while on treatment. Pvalues (vs. Erlotinib)=0.0001.

FIG. 2. Compound D prevents acquired resistance to Erlotinib and leadsto regression of Erlotinib-resistant tumors in mice implanted with atumor from a Head & Neck (H&N) cancer patient. Mice were treated with(a) vehicle (⋄); (b) Erlotinib (□); (c) Compound D (Δ); or (d)Erlotinib+Compound D (∘). Treatment with Erlotinib (group b) initiallyled to tumor regression. While on treatment, the tumors developedresistance to Erlotinib and regrew. Combined treatment of Erlotinib andCompound D induced tumor regression and none of the tumors regrew (groupd), consistent with the results displayed in FIG. 1. Followingresistance to Erlotinib has been acquired, the mice in group b whosetumors were ˜130 mm³ on day 10 were divided into two groups, the firstremained on Erlotinib alone (□) and the second received a combinedtreatment of Erlotinib+Compound D starting on day 10 of treatment (●).While tumors significantly grew under treatment with Erlotinib alone(□), the combined treatment of Compound D and Erlotinib induced tumorregression (●).

FIG. 3. Compound D prevents acquired resistance to Erlotinib in largetumors (700 mm³). Mice were treated with vehicle (⋄), Erlotinib (□),Compound D (Δ), or Erlotinib+Compound D (∘). Treatments were initiatedwhen average tumor size was ˜700 mm³.

FIG. 4. Combined treatment with Afinitor and Compound D induces tumorregression of aggressive Uteral AdenoSarcoma in mice. Mice implantedwith patient-derived xenografts of violent Uteral AdenoSarcoma weretreated when average tumor size was ˜130 mm³ with either vehicle (⋄),Afinitor (□), Compound D (Δ), or Afinitor+Compound D (∘). The averagetumor volume of the Afinitor-treated group (□) indicated tumor growthinhibition. While Compound D (Δ) alone had no significant effect ontumor growth as compared to the control, the combined treatment withAfinitor+Compound D (∘) led to tumor regression. In alternative analysisof the results (demonstrated in FIG. 5)—while none of the mice respondedto Compound D alone (Δ) and in the Afinitor-treated group (□) half ofthe group responded and the other half did not, the combined treatmentwith Afinitor+Compound D (∘) led to positive response of all mice in thegroup.

FIGS. 5A-5C. Compound D prevents acquired resistance to Afinitor (A) andleads to regression of Afinitor-resistant tumors (B). Mice implantedwith patient-derived xenografts of aggressive Uteral AdenoSarcoma, werefirst treated when tumors were ˜130 mm³ as described in FIG. 4. Micewere treated with either vehicle (diamonds); Afinitor (squares);Compound D (triangles); or Afinitor+Compound D (circles). FIG. 5A. TheAfinitor-treated group was divided to responders (open squares, group A,n=8) vs. non-responders (grey squares, group B, n=7). Afinitor treatmentof group A initially induced tumor regression, but while on treatmentall tumors developed resistance to Afinitor and aggressively progressed.Combined treatment of Afinitor and Compound D from day0 (treatmentinitiation) induced tumor regression and their average tumor volumeremained low till the end of the experiment (∘). FIG. 5B. Followingresistance to Afinitor has been acquired, the mice of group A weredivided into two groups, the first remained on Afinitor alone (□) andthe second received combined treatment of Afinitor+Compound D startingon day 6 of treatment (●). While tumors significantly progressed undertreatment with Afinitor alone (□), the combined treatment of Compound Dand Afinitor induced tumor regression (●). The graph in FIG. 5Brepresents growth rates in %, while the 100% for each tumor was definedas its volume at day 6. FIG. 5C Combined treatment withAfinitor+Compound D of highly aggressive Uterine Adenosarcoma cancerwith no available medical treatment delayed acquired resistance toAfinitor and achieved complete response in 40% of the group.

FIGS. 6A-6E. Dual modulators of IRS/Stat3 potently inhibit Stat3phosphorylation in intact cells in a dose-dependent manner, and theirinhibitory effect on Stat3 lasts long after the modulators are washedout the cells. A. Human melanoma A375 cells were treated with theindicated concentrations of compounds A or D for 1.3 and 4.5 hr. Cellswere lysed and immunoblotted with antibodies against phospho-Y705 Stat3(pSTAT3) and Stat3. A dose response inhibition of pStat3 withsub-micromolar IC50 values is demonstrated, and the effect ispotentiated in time. B. Cells were treated with 2 μM Compound A andlysed following the indicated times. C. A375 cells were treated withcompound D for 4 hr, then washed with medium several times and lysedafter 4, 24 and 48 hr of incubation without inhibitors. D.Dose-dependent inhibition of pStat3 showing IC50 values of <1 μM forcompounds A, C, D; 1 μM for compound B and 2 μM for compound F. E.Complete inhibition of Stat3 Y705-phosphorylation in melanoma A375 cells24 hr post treatment with 3 μM compounds IV-1, IV-2, IV-3 and IV-4.

FIGS. 7A-7E. Acquired resistance to BRAF inhibitors (BRAFi) in melanomais accompanied by enhanced Stat3 phosphorylation levels, and treatmentwith BRAFi of human melanoma cells surprisingly induces a dramaticincrease in pStat3. FIG. 7A. Highly increased levels of pStat3 in theBRAFi-resistant melanoma clone (R) as compared to the parental melanomacells (P). Human metastatic melanoma 451-Lu cells (P) and thePLX4032-resistant clone, both possess mutated BRAF, were grown in aserum-free medium and immunoblotted with anti-pStat3 Ab followed byanti-Stat3 Ab. FIG. 7B. Highly increased levels of pStat3 in melanomacells with mutated BRAF, derived from patients that acquired resistanceto the BRAFi PLX4032 (R) compared to melanoma cells with mutated BRAFfrom naive patients that were not treated with PLX4032 (N). Cells frompatients were grown in complete medium and immunoblotted as describedabove. FIGS. 7C-E. Treatment with PLX4032 of BRAF-mutated human melanomacells surprisingly induces a dramatic increase in pStat3. Compounds A/Dblock the basal and the PLX4032-induced pStat3. Treatment ofPLX4032-sensitive human melanoma A375 (C), 451-Lu (D) or Mel1617 (E)cells with 1 μM PLX4032 for 18-24 hr induces a dramatic increase inpStat3. Compounds A (FIG. 7C, D) or D (FIG. 7E) block both the basal andthe PLX4032-induced pStat3. OSI-906, an ATP-competitive inhibitor ofIGF1R, has no effect on pStat3.

FIGS. 8A-8D. Dual modulators of IRS/Stat3 efficiently inhibit pStat3 inBRAFi-resistant melanoma cells, which acquired resistance in culture orin patients. Their ability to inhibit pStat3 was exemplified in variouscancer types. FIG. 8A-B. Compound A, as opposed to the IGF1R inhibitorOSI-906, potently inhibits pStat3 in BRAFi-resistant clones of melanoma,451-BR (FIG. 8A) and Mel1617-BR (FIG. 8B). FIG. 8C. Compound A, and morepotently compound D, inhibit pStat3 in melanoma cells from patients (iand ii) who acquired resistance to PLX4032 following treatment. FIG. 8D.Cells of various cancer types were treated with compound D for 4 hr inserum-free medium, and 20 hr in medium with or without 10% serum.Asterisks indicate 10 μM concentration for RPMI8226 and HepG2 cells.

FIGS. 9A-9B. Treatment of melanoma A375 cells with Compound A inducesPBMC's chemotaxis. A375 cells were treated with the indicatedconcentrations of Compound A and washed twice with the medium 4 hrsafter treatment where indicated (Wash). 30 hrs following treatment thecell medium was transferred to lower plate of chemotaxis device. 10,000PBMCs/well were added to the upper plate. In addition, PBMCs were addedinto lower plate as positive control. FIG. 9A. Chemotaxis of PBMCstowards Compound A was examined 24 hrs later by Cell Titer Glo analysisof the lower plate. FIG. 9B. PBMC calibration curve of 10-10,000cells/well.

FIG. 10. Combined treatment of Cetuximab±Afatinib with Compound D showsa dramatic delay in tumor recurrence compared to Cetuximab±Afatinibalone in mice implanted with a tumor from a HNSCC patient. Mice weretreated for 9 days with (a) vehicle (⋄); (b) Compound D (Δ); (c)Cetuximab (□); (d) Cetuximab+Afatinib (┘); (e) Cetuximab+Compound D (∘);or (e) Cetuximab+Afatinib+Compound D (●). Treatments were initiated whenaverage tumor size was ˜110 mm³. Combined treatment of Compound D witheither Cetuximab or Cetuximab+Afatinib for 9 days only, significantlydelayed recurrence of regressed tumors and prolonged response toCetuximab or to Cetuximab+Afatinib.

FIG. 11. Compound D synergizes with the combination of mutated-BRAFinhibitor (BRAFi) and MEK inhibitor (MEKi), to induce dramatic tumorregression in mice implanted with tumor cells from a melanoma patientwho has acquired resistance to mutated-BRAF inhibitory drug treatment.Mice were treated with (a) vehicle (⋄); (b) Trametinib(MEKi)+Vemurafenib (BRAFi) (□); (c) Compound D (Δ); or (d)Trametinib+Vemurafenib+Compound D (∘). Treatments were initiated whenaverage tumor size was ˜60 mm³. Tumors aggressively progressed in allmice treated with Trametinib+Vemurafenib, while combined treatment ofTrametinib+Vemurafenib with Compound D induced tumor regression in allmice in this group and none of them regrew while on treatment. P valuesof Trametinib+Vemurafenib+Compound D vs. Trametinib+Vemurafenib=0.0001.

FIG. 12. Compound D synergizes with MEK inhibitor Trametinib to inducetumor regression in mice implanted with tumor from Adenoid CycticCarcinoma patient harboring mutation in BRAF. Mice were treated with (a)vehicle (⋄); (b) Trametinib (□); (c) Compound D (Δ); or (d)Trametinib+Compound D (∘). Treatments were initiated when average tumorsize was ˜65 mm³. Treatment with Trametinib induced tumor regression,but while on treatment tumors progressed. Combined treatment ofTrametinib and Compound D induced tumor regression and none of thesetumors regrew while on treatment. The study included two phases oftreatments day0-day13 and day24-day31.

FIGS. 13A-13B. Compound D re-sensitizes Gemcitabine-resistant tumors toGemcitabine in mice implanted with tumor from a liver metastasis of apancreatic cancer patient. A. Mice were treated with Gemcitabine for 35days and a week later regressed tumors acquired resistance toGemcitabine and progressed. At this point when average tumor size wasalready ˜110 mm³ the Gemcitabine-treated mice were divided to twogroups: (a) Gemcitabine (□); (b) Gemcitabine+Compound D (∘). While alltumors treated with Gemcitabine progressed, combined treatment withCompound D+Gemcitabine led to tumor regression in half of the group andsignificant tumor growth inhibition in terms of average tumor size ofthe group compared to the Gemcitabine-treated group (p value=7.35*10⁻⁵).B. At the end of the experiment, tumor pieces, similar in size, werecultured in plates (3 tumors per group) to test their viability andproliferative activity. Nine days later the plates were fixed andstained, showing massive proliferation in the Gemcitabine-treated tumorsas opposed to a very low to negligible proliferative activity in thetumors from mice treated with Gemcitabine+Compound D.

FIG. 14. Compound D prevents acquired resistance to Cetuximab in miceimplanted with a tumor from an adnexal adeno carcinoma metastaticpatient. Mice were treated for 32 days with (a) vehicle (⋄); (b)Compound D (Δ); (c) Cetuximab (□); (d) Cetuximab+Compound D (∘).Treatments were initiated when average tumor size was ˜90 mm³. Treatmentwith Cetuximab led to transient tumor growth attenuation followed byacquired resistance to Cetuximab, while the combined treatment ofCetuximab+Compound D induced tumor regression and prevented acquiredresistance to Cetuximab.

FIG. 15. Compound D prevents acquired resistance to the combinedtreatment of Cetuximab and FOLFIRI (an approved treatment for coloncancer patients) in mice implanted with a tumor from a colon cancerpatient. Mice were treated for 14 days with (a) vehicle (⋄); (b)Compound D (Δ); (c) Cetuximab+FOLFIRI (□); (d)Cetuximab+FOLFIRI+Compound D (∘); (e) FOLFIRI (▪); and (f) Cetuximab (▪,dashed line). Treatments were initiated when average tumor size was ˜110mm³. Combined treatment of Cetuximab+FOLFIRI with or without Compound Dinduced tumor regression at the first week of treatment. While alltumors in mice treated with Cetuximab+FOLFIRI developed resistance tothe treatment during the second week of treatment and aggressivelyprogressed, the combined treatment with Compound D prevented acquiredresistance to Cetuximab+FOLFIRI.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

The present invention relates to the treatment of cancer usingcombination therapy comprising a dual modulator of Insulin ReceptorSubstrate (IRS) and signal transducer and activator of transcription 3(Stat3), in combination with (i) a modulator of a protein kinase (PK)selected from an Epidermal Growth Factor inhibitor (EGFR inhibitor) andEGFR antibody; (ii) an inhibitor of mammalian target of rapamycin(mTOR); (iii) a mitogen-activated protein kinase (MEK) inhibitor; (iv) amutated B-Raf inhibitor; (v) a chemotherapeutic agent like Gemcitabine,5-FU, Irinotecan and Oxaliplatin; and (vi) certain combinations thereof.The combination can be used to treat a tumor that has developedresistance to an EGFR inhibitor, EGFR antibody, mTOR inhibitor, MEKinhibitor, mutated B-Raf inhibitor, chemotherapeutic agents, and certaincombinations thereof, or to prevent acquired resistance of a tumor toany of said inhibitors or agents, or to prevent tumor recurrencefollowing cease of treatment with any of said inhibitors or agents or acombination thereof. The combination provides a therapeutic effect whichis at least additive, and is preferably synergistic. The presentinvention further relates to the treatment of cancer using combinationtherapy comprising a dual modulator of IRS and Stat3, in combinationwith an immunotherapy agent. The combination can be used to sensitize atumor to immunotherapy.

Insulin Receptor Substrate (IRS)/Signal Transducer and Activator ofTranscription 3 (Stat3) Dual Modulators

Any compound of the general structure of formula (I) can be used in thecompositions of the present invention:

wherein

R¹, R², R⁵ and R⁶ are each independently selected from H, C₁-C₄ alkyl,C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₄ alkyl-C₂-C₆ alkenyl, C₁-C₄alkyl-C₂-C₆ alkynyl, (CH₂CH₂O)_(n)H, C₃-C₇ cycloalkyl, aryl,heterocyclyl, heteroaryl, (C₁-C₄)-alkylaryl, (C₁-C₄)-alkylheterocyclyl,(C₁-C₄)-alkylheteroaryl, haloalkyl, acyl and a functional group thatgives rise to hydroxyl upon hydrolysis;

R³, R⁴, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³ and R¹⁴ are each independentlyselected from H, C₁-C₄ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₄alkyl-C₂-C₆ alkenyl, C₁-C₄ alkyl-C₂-C₆ alkynyl, C₃-C₇ cycloalkyl, aryl,heterocyclyl, heteroaryl, (C₁-C₄)-alkylaryl, (C₁-C₄)-alkylheterocyclyl,(C₁-C₄)-alkylheteroaryl, halogen, haloalkyl, NO₂, CN, N₃, SO₂R^(a),COOR^(a), CSNR^(a)R^(b), CSOR^(a), OR^(a), CONR^(a)R^(b), NR^(a)R^(b),SR^(a), and CH₂SR^(a), wherein R^(a) and R^(b) are each independently H,C₁-C₄ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₄ alkyl-C₂-C₆ alkenyl,C₁-C₄ alkyl-C₂-C₆ alkynyl, C₃-C₇ cycloalkyl, aryl, heterocyclyl,heteroaryl, (C₁-C₄)-alkylaryl, (C₁-C₄)-alkylheterocyclyl,(C₁-C₄)-alkylheteroaryl, haloalkyl, (CH₂CH₂O)_(n)H, acyl or a functionalgroup that gives rise to hydroxyl upon hydrolysis; and

R¹⁵ is H, C₁-C₄ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₄ alkyl-C₂-C₆alkenyl, C₁-C₄ alkyl-C₂-C₆ alkynyl, haloalkyl, or OR^(b) wherein R^(b)is independently H or C₁-C₄ alkyl;

including salts, hydrates, solvates, polymorphs, optical isomers,geometrical isomers, enantiomers, diastereomers, and mixtures thereof.Each possibility represents a separate embodiment of the invention.

In an exemplary embodiment, the compound is a compound represented byformula A:

In another exemplary embodiment, the compound is a compound representedby formula B:

In another exemplary embodiment, the compound is a compound representedby formula C:

In another exemplary embodiment, the compound is a compound representedby formula D:

In another exemplary embodiment, the compound is a compound representedby formula E:

In another exemplary embodiment, the compound is a compound representedby formula F:

In another exemplary embodiment, the compound is a compound representedby formula G:

In another exemplary embodiment, the compound is a compound representedby formula H:

In another exemplary embodiment, the compound is a compound representedby formula I:

In another exemplary embodiment, the compound is a compound representedby formula J:

In another embodiment, the compound is a compound of formula I whereinR¹, R², R⁴, R⁵, R⁶, R¹⁰, R¹², R¹³, R¹⁴ and R¹⁵ are each H; R⁷ is OH; andat least one of R³, R⁸, R⁹ and R¹¹ is halogen.

In another embodiment, the compound is a compound of formula I whereinR¹, R², R⁴, R⁵, R⁶, R⁸, R¹⁰, R¹², R¹³, R¹⁴ and R¹⁵ are each H; R⁷ is OH;and at least one of R³, R⁹ and R¹¹ is halogen.

In another embodiment, the compound is a compound of formula I whereinR¹, R², R⁵ and R⁶ are each H or a functional group that gives rise tohydroxyl upon hydrolysis.

In another embodiment, the compound is a compound of formula I whereinR⁷ is H or OR^(a) and R¹, R², R⁵, R⁶, and R^(a) are each H or afunctional group that gives rise to hydroxyl upon hydrolysis.

In another embodiment, the compound is a compound of formula I whereinR¹³ and R¹⁴ are each independently H, C₁-C₄ alkyl, C₂-C₆ alkenyl, C₂-C₆alkynyl, C₁-C₄ alkyl-C₂-C₆ alkenyl or C₁-C₄ alkyl-C₂-C₆ alkynyl.

In another embodiment, the compound is a compound of formula I whereinat least one of R¹³ and R¹⁴ is H or C₁-C₄ alkyl.

In another embodiment, the compound is a compound of formula I whereinR³, R⁴, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ are each independentlyH, halogen, haloalkyl, OH, NO₂, CN, or CH₂SR^(a), wherein R^(a) is asdefined hereinabove.

In another embodiment, the compound is a compound of formula I whereinR⁴ is H.

In another embodiment, the compound is a compound of formula I whereinR⁴ is CN.

In another embodiment, the compound is a compound of formula I whereinR⁴, R¹¹, R¹², R¹³, R¹⁴ and R¹⁵ are each H.

In another embodiment, the compound is a compound of formula I whereinR¹³, R¹⁴ and R¹⁵ are each H.

In another embodiment, the compound is a compound of formula I whereinR³, R⁷, R⁸, R⁹, R¹⁰ and R¹¹ are each independently H, halogen,haloalkyl, CH₂SR^(a) or OH; R⁴, R¹², R¹³ and R¹⁴ are each independentlyH, C₁-C₄ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₄ alkyl-C₂-C₆ alkenyl,C₁-C₄ alkyl-C₂-C₆ alkynyl, aryl, halogen, haloalkyl, NO₂, or CN; and R¹⁵is H.

In another embodiment, the compound is a compound of formula I whereinR³, R⁷, R⁸, R⁹, R¹⁰ and R¹¹ are each independently H, halogen,haloalkyl, OH or CH₂SR^(a); and R⁴, R¹², R¹³, R¹⁴ and R¹⁵ are each H, ora C₁-C₄ alkyl.

In another embodiment, the compound is a compound of formula I whereinR¹, R², R⁵ and R⁶ are each H or a functional group that gives rise tohydroxyl upon hydrolysis; R³, R⁸, and R⁹ are each independently H,halogen, haloalkyl, or CH₂SR^(a); R⁷, R¹⁰ and R¹¹ are each independentlyH, halogen, haloalkyl, OH or a functional group that gives rise tohydroxyl upon hydrolysis; and R⁴, R¹², R¹³, R¹⁴ and R¹⁵ are each H, orC₁-C₄ alkyl.

In another embodiment, the compound is a compound of formula I whereinthe compound is represented by any one of the structures:

Each possibility represents a separate embodiment of the presentinvention.

In other embodiments, the compound is a compound represented by thestructure of formula II:

wherein

-   -   R¹, R², R⁵ and R⁶ are independently selected from H, C₁-C₄        alkyl, acyl and a functional group that gives rise to hydroxyl        upon hydrolysis;    -   R³ and R⁷ are independently selected from H, halogen, haloalkyl        and OR⁸ wherein R⁸ is H, C₁-C₄ alkyl, acyl or a functional group        that gives rise to hydroxyl upon hydrolysis; and    -   R⁴ is H or CN,        -   including salts, hydrates, solvates, polymorphs, optical            isomers, geometrical isomers, enantiomers, diastereomers,            and mixtures thereof.

In other embodiments, the compound is a compound represented by thestructure of formula II, wherein

-   -   R¹, R², R⁵ and R⁶ are independently selected from H, C₁-C₄        alkyl, (CH₂CH₂O)_(n)H wherein n is an integer of 1 to 20, acyl        and a functional group that gives rise to hydroxyl upon        hydrolysis;    -   R³ and R⁷ are independently selected from H, halogen, C₁-C₄        alkyl, haloalkyl and OR¹⁶ wherein R¹⁶ is H, C₁-C₄ alkyl,        (CH₂CH₂O)_(n)H, acyl or a functional group that gives rise to        hydroxyl upon hydrolysis; and    -   R⁴ is H or CN,    -   including salts, hydrates, solvates, polymorphs, optical        isomers, geometrical isomers, enantiomers, diastereomers, and        mixtures thereof.

Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, the compound is a compound of formula II whereinR⁴ is CN.

In other embodiments, the compound is a compound of formula II whereinR¹, R², R⁵ and R⁶ are each hydrogen.

In other embodiments, the compound is a compound of formula II whereinR¹, R², R⁵ and R⁶ are each CH₃.

In other embodiments, the compound is a compound of formula II whereinR³ and R⁷ are each a hydrogen, halogen, halomethyl, OH or OCH₃.

In other embodiments, the compound is a compound of formula II whereinR¹, R², R⁵ and R⁶ are each H, R³ is halogen and R⁷ is OH.

In other embodiments, the compound is a compound of formula II whereinR¹, R², R⁵ and R⁶ are each H, and R³ and R⁷ are each halogen.

In other embodiments, the compound is a compound of formula II whereinR¹, R², R⁵ and R⁶ are each H, R³ is halomethyl and R⁷ is OH.

In other embodiments, the compound is a compound of formula II whereinR¹, R², R⁵ and R⁶ are each H, R³ is halogen and R⁷ is H.

In other embodiments, the compound is a compound of formula II whereinR¹, R², R⁵ and R⁶ are each H, R³ is OH and R⁷ is halogen.

In other embodiments, the compound is a compound of formula II whereinR¹, R², R⁵ and R⁶ are each CH₃, R³ is halogen and R⁷ is OCH₃.

In other embodiments, the compound is a compound of formula II whereinR¹, R², R⁵ and R⁶ are each CH₃, and R³ and R⁷ are each halogen.

In other embodiments, the compound is a compound of formula II whereinR⁴ is hydrogen.

In other embodiments, the compound is a compound of formula II whereinR¹, R², R⁵ and R⁶ are each hydrogen.

In other embodiments, the compound is a compound of formula II whereinR¹, R², R⁵ and R⁶ are each CH₃.

In other embodiments, the compound is a compound of formula II whereinR³ and R⁷ are each hydrogen, halogen, halomethyl, OH or OCH₃.

In other embodiments, the compound is a compound of formula II whereinR¹, R², R⁵ and R⁶ are each H, R³ is halogen and R⁷ is OH.

In other embodiments, the compound is a compound of formula II whereinR¹, R², R⁵ and R⁶ are each H, and R³ and R⁷ are each halogen.

In other embodiments, the compound is a compound of formula II whereinR¹, R², R⁵ and R⁶ are each H, R³ is halomethyl and R⁷ is OH.

In other embodiments, the compound is a compound of formula II whereinR¹, R², R⁵ and R⁶ are each H, R³ is halogen and R⁷ is H.

In other embodiments, the compound is a compound of formula II whereinR¹, R², R⁵ and R⁶ are each H, R³ is OH and R⁷ is halogen.

In other embodiments, the compound is a compound of formula II whereinR³ is halogen and R⁷ is OCH3.

In other embodiments, the compound is a compound of formula II whereinR¹, R², R⁵ and R⁶ are each CH₃, and R³ and R⁷ are each halogen.

In other embodiments, the compound of formula (II) is represented by anyof the following compounds:

Each possibility represents a separate embodiment of the presentinvention. In another embodiment, the compound is represented by thestructure of formula III:

wherein

-   -   R¹, R², R⁵ and R⁶ are independently selected from H, C₁-C₄        alkyl, (CH₂CH₂O)_(n)H wherein n is an integer of 1 to 20, acyl        and a functional group that gives rise to hydroxyl upon        hydrolysis;    -   R³, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³ and R¹⁴ are independently        selected from H, halogen, C₁-C₄ alkyl, haloalkyl and OR¹⁶        wherein R¹⁶ is H, C₁-C₄ alkyl, (CH₂CH₂O)_(n)H, acyl or a        functional group that gives rise to hydroxyl upon hydrolysis;        and    -   R⁴ is H or CN.

In other embodiments, the compound is represented by the structure offormula IV:

wherein

A is H or CN;

Z is S, SO or SO₂;

X¹, X², X³, X⁴, X⁵, Y¹ and Y² are each independently selected from H,halogen, alkyl, haloalkyl and OR¹; and

Y³ and Y⁴ are each OR¹, wherein each IV is independently H, C₁-C₄ alkyl,—(CH₂CH₂O)_(n)H wherein n is an integer of 1 to 20, acyl or a functionalgroup that gives rise to hydroxyl upon hydrolysis, including salts,hydrates, solvates, polymorphs, optical isomers, geometrical isomers,enantiomers, diastereomers, and mixtures thereof.

In some embodiments, the compound is a compound of formula IV wherein Ais H.

In other embodiments, the compound is a compound of formula IV wherein Ais CN.

In other embodiments, the compound is a compound of formula IV wherein Zis S.

In other embodiments, the compound is a compound of formula IV wherein Zis SO₂.

In other embodiments, the compound is a compound of formula IV whereinat least one of X¹, X², X³, X⁴, Y¹ and Y² is a halogen.

In other embodiments, the compound is a compound of formula IV whereinat least one of X¹, X², X³, X⁴, Y¹ and Y² is Br.

In other embodiments, the compound is a compound of formula IV whereinat least one of X¹, X², X³, X⁴, Y¹ and Y² is I.

In other embodiments, the compound is a compound of formula IV whereinX¹, X², X³, and X⁴ are each selected from H or a halogen, wherein thehalogen is preferably Br or I.

In other embodiments, the compound is a compound of formula IV whereinX² is H.

In other embodiments, the compound is a compound of formula IV whereinX⁵ is H.

In other embodiments, the compound is a compound of formula IV whereinX⁵ is alkyl, preferably methyl.

In other embodiments, the compound is a compound of formula IV whereinY³ and Y⁴ are each OH.

In other embodiments, the compound is a compound of formula IV whereinY¹ and Y² are each OH.

In other embodiments, the compound is a compound of formula IV wherein Ais H, Z is S, Y³ and Y⁴ are each OH, and X¹ is a halogen selected fromBr and I.

Each possibility represents a separate embodiment of the presentinvention.

In other embodiments, the compound of formula (IV) is represented by anyof the following compounds:

A currently preferred compound of formula IV is a compound of formulaIV-4.

In other embodiments, the compound is any of the derivatives describedin A) PCT International Patent Application Publication No. WO2008/068751; B) PCT International Patent Application Publication No. WO2009/147682; or C) PCT International patent Application No. WO2012/090204. The contents of each of the aforementioned references areincorporated by reference herein in their entirety as if fully set forthherein.

It is understood that all conformers, geometrical isomers,stereoisomers, enantiomers and diastereomers of any of the compoundsdescribed herein, are encompassed and may be used in the combinationsand methods described by the present application.

Without being bound to any particular theory or mechanism of action, itis contemplated that the compounds of the present invention areinhibitors of PK signaling, such as IGF-1R. It has now been surprisinglyfound that these compounds, in addition to being inhibitors of IGF-1R,also lead to the dissociation of the IGF-1R substrates IRS1/2 from thecell membrane, inhibitory serine phosphorylation and/or degradation ofthe IRS1/2 proteins. This activity leads to long lasting inhibition ofthe IGF-1R and IR pathways, growth inhibition of a wide range of cancercell types, and potent anti-tumor effects. These compounds are thereforereferred to as “modulators of IRS”. Thus, in another embodiment, thepresent invention provides a method of inhibiting, treating orpreventing an insulin-like growth factor 1 receptor (IGF-1R) and/orinsulin receptor substrate 1 (IRS1) and/or insulin receptor substrate 2(IRS2) signaling related disorder in a subject comprising the step ofadministering to the subject a pharmaceutical composition comprising atherapeutically effective amount of at least one compound represented bythe structure of formula I or any of the compounds covered by suchformula, together with an anti-cancer agent selected from EGFRinhibitor, EGFR antibody, mTOR inhibitor and/or immunotherapy agent,wherein the compound of formula I and the anti-cancer agent togetherprovide an anti-cancer effect which is at least additive, and ispreferably synergistic. In some embodiments, the compound of formula Iis an inhibitor of an insulin receptor or an insulin-like growthfactor-1 receptor (IGF-1R) signaling, and/or the compound of formula Iinteracts with, affects or inhibits a substrate protein in the IGF-1Rmediated pathway. In some embodiments, the substrate protein is InsulinReceptor Substrate 1 (IRS1), Insulin Receptor Substrate 2 (IRS2), or acombination thereof. In one particular embodiment, the compound offormula I is an IGF-1R kinase inhibitor that leads to at least one ofthe dissociation of IRS1 or IRS2 from the cell membrane, phosphorylationof IRS1 or IRS2, and/or degradation of IRS1 or IRS2, in any order.

IGF1R and specifically IRS1 are one of the key mechanisms for resistanceto EGFR inhibition (Buck E. et al. Feedback mechanisms promotecooperativity for small molecule inhibitors of epidermal andinsulin-like growth factor receptors. Cancer Res. 2008 Oct. 15;68(20):8322-32).

The compounds described herein are also modulators of signal transducerand activator of transcription 3 (Stat3). In some embodiments, thecompounds lead to the inhibition of Stat3 phosphorylation in cancercells. Increased levels of Stat3 phosphorylation are detected in variouscancers and drug-resistant cancers, leading to enhanced cancer survival.Without wishing to be bound by any particular theory or mechanism ofaction it is contemplated that inhibiting Stat3 activity may synergizewith such PK inhibitor drugs, which as a side effect upregulate Stat3,may prevent acquired resistance to such drugs and may be effective fordrug-resistant cancers. Furthermore, Stat3 is often activated in cancerand directly involved in the implementation and maintenance of thecancer immunosuppressive microenvironment and plays a central role intumor immune evasion. Without wishing to be bound by any particulartheory or mechanism of action, it is contemplated that inhibition ofStat3 phosphorylation un-masks the tumor from the local immune systemand sensitize them to immunotherapy e.g. antibodies against PDLs, PD1,CTLA4 or any other immunotherapy agents.

Chemical Definitions

An “alkyl” group refers to any saturated aliphatic hydrocarbon,including straight-chain and branched-chain alkyl groups. In oneembodiment, the alkyl group has 1-12 carbons designated here asC₁-C₁₂-alkyl. In another embodiment, the alkyl group has 1-6 carbonsdesignated here as C₁-C₆-alkyl. In another embodiment, the alkyl grouphas 1-4 carbons designated here as C₁-C₄-alkyl. The alkyl group may beunsubstituted or substituted by one or more groups selected fromhalogen, hydroxy, alkoxy carbonyl, amido, alkylamido, dialkylamido,nitro, amino, alkylamino, dialkylamino, carboxyl, thio and thioalkyl.

An “alkenyl” group refers to an aliphatic hydrocarbon group containingat least one carbon-carbon double bond including straight-chain,branched-chain and cyclic alkenyl groups. In one embodiment, the alkenylgroup has 2-8 carbon atoms designated here as C₂-C₈-alkenyl. In anotherembodiment, the alkenyl group has 2-6 carbon atoms in the chaindesignated here as C₂-C₆-alkenyl. Exemplary alkenyl groups includeethenyl, propenyl, n-butenyl, i-butenyl, 3-methylbut-2-enyl, n-pentenyl,heptenyl, octenyl, cyclohexyl-butenyl and decenyl. The alkenyl group canbe unsubstituted or substituted through available carbon atoms with oneor more groups defined hereinabove for alkyl.

An “alkynyl” group refers to an aliphatic hydrocarbon group containingat least one carbon-carbon triple bond including straight-chain andbranched-chain. In one embodiment, the alkynyl group has 2-8 carbonatoms in the chain designated here as C₂-C₈-alkynyl. In anotherembodiment, the alkynyl group has 2-6 carbon atoms in the chaindesignated here as C₂-C₆-alkynyl. Exemplary alkynyl groups includeethynyl, propynyl, n-butynyl, 2-butynyl, 3-methylbutynyl, n-pentynyl,heptynyl, octynyl and decynyl. The alkynyl group can be unsubstituted orsubstituted through available carbon atoms with one or more groupsdefined hereinabove for alkyl.

The term “C₃-C₇ cycloalkyl” used herein alone or as part of anothergroup refers to any saturated or unsaturated (e.g., cycloalkenyl,cycloalkynyl) monocyclic or polycyclic group. Non-limiting examples ofcycloalkyl groups are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexylor cycloheptyl. Non-limiting examples of cycloalkenyl groups includecyclopentenyl, cyclohexenyl and the like. The cycloalkyl group can beunsubstituted or substituted with any one or more of the substituentsdefined above for alkyl. Similarly, the term “cycloalkylene” means abivalent cycloalkyl, as defined above, where the cycloalkyl radical isbonded at two positions connecting together two separate additionalgroups.

The term “aryl” used herein alone or as part of another group refers toan aromatic ring system containing from 6-14 ring carbon atoms. The arylring can be a monocyclic, bicyclic, tricyclic and the like. Non-limitingexamples of aryl groups are phenyl, naphthyl including 1-naphthyl and2-naphthyl, and the like. The aryl group can be unsubstituted orsubstituted through available carbon atoms with one or more groupsdefined hereinabove for alkyl.

The term “heteroaryl” used herein alone or as part of another grouprefers to a heteroaromatic system containing at least one heteroatomring wherein the atom is selected from nitrogen, sulfur and oxygen. Theheteroaryl contains 5 or more ring atoms. The heteroaryl group can bemonocyclic, bicyclic, tricyclic and the like. Also included in thisdefinition are the benzoheterocyclic rings. If nitrogen is a ring atom,the present invention also contemplates the N-oxides of the nitrogencontaining heteroaryls. Non-limiting examples of heteroaryls includethienyl, benzothienyl, 1-naphthothienyl, thianthrenyl, furyl,benzofuryl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl,pyrimidinyl, pyridazinyl, indolyl, isoindolyl, indazolyl, purinyl,isoquinolyl, quinolyl, naphthyridinyl, quinoxalinyl, quinazolinyl,cinnolinyl, pteridinyl, carbolinyl, thiazolyl, oxazolyl, isothiazolyl,isoxazolyl and the like. The heteroaryl group can be unsubstituted orsubstituted through available atoms with one or more groups definedhereinabove for alkyl.

The term “heterocyclic ring” or “heterocyclyl” used herein alone or aspart of another group refers to a five-membered to eight-membered ringsthat have 1 to 4 heteroatoms, such as oxygen, sulfur and/or nitrogen, inparticular nitrogen, either alone or in conjunction with sulfur oroxygen ring atoms. These five-membered to eight-membered rings can besaturated, fully unsaturated or partially unsaturated, with fullysaturated rings being preferred. Preferred heterocyclic rings includepiperidinyl, pyrrolidinyl pyrrolinyl, pyrazolinyl, pyrazolidinyl,morpholinyl, thiomorpholinyl, pyranyl, thiopyranyl, piperazinyl,indolinyl, dihydrofuranyl, tetrahydrofuranyl, dihydrothiophenyl,tetrahydrothiophenyl, dihydropyranyl, tetrahydropyranyl,dihydrothiazolyl, and the like. The heterocyclyl group can beunsubstituted or substituted through available atoms with one or moregroups defined hereinabove for alkyl.

The term “acyl” as used herein encompasses groups such as, but notlimited to, formyl, acetyl, propionyl, butyryl, pentanoyl, pivaloyl,hexanoyl, heptanoyl, octanoyl, nonanoyl, decanoyl, undecanoyl,dodecanoyl, benzoyl and the like. Currently preferred acyl groups areacetyl and benzoyl.

A “hydroxy” group refers to an OH group. An “alkoxy” group refers to an—O-alkyl group wherein R is alkyl as defined above.

A “thio” group refers to an —SH group. An “alkylthio” group refers to an—SR group wherein R is alkyl as defined above.

An “amino” group refers to an NH₂ group. An alkylamino group refers toan —NHR group wherein R is alkyl is as defined above. A dialkylaminogroup refers to an —NRR′ group wherein R and R′ are alkyl as definedabove.

An “amido” group refers to a —C(O)NH₂ group. An alkylamido group refersto an —C(O)NHR group wherein R is alkyl is as defined above. Adialkylamido group refers to an —C(O)NRR′ group wherein R and R′ arealkyl as defined above.

A “thioamide” group refers to a —C(S)NHR group, where R is either alkyl,aryl, alkylaryl or H.

A “polyoxyalkylene” group refers to a (CH₂CH₂O)_(n)H group whereinn=1-20. Currently preferred polyoxyalkylene groups arepolyethyleneglycol (PEG) and polypropyleneglycol.

The term “halogen” or “halo” as used herein alone or as part of anothergroup refers to chlorine, bromine, fluorine, and iodine. The term“haloalkyl” refers to an alkyl group having some or all of the hydrogensindependently replaced by a halogen group including, but not limited to,trichloromethyl, tribromomethyl, trifluoromethyl, triiodomethyl,difluoromethyl, chlorodifluoromethyl, pentafluoroethyl,1,1-difluoroethyl bromomethyl, chloromethyl, fluoromethyl, iodomethyl,and the like.

Examples of functional groups that give rise to hydroxyl upon hydrolysisinclude, but are not limited to, esters, anhydrides, carbamates,carbonates and the like. For example, when any of R¹, R², R⁵ or R⁶ is anacyl group (COR), the resulting functional group is an ester (OCOR).When any of R¹, R², R⁵ or R⁶ is an amide group (CONHR), the resultingfunctional group is a carbamate (OCONHR). When any of R¹, R², R⁵ or R⁶is a carboxylate group (COOR), the resulting functional group is acarbonate (OCOOR).

Within the scope of the present invention are prodrugs of the compoundsdisclosed herein. The term “prodrug” represents compounds which arerapidly transformed in vivo to any of compounds represented by formulaI, for example by hydrolysis in the blood. Thus, the term “prodrug”refers to a precursor of any of the compounds of the present inventionthat is pharmaceutically acceptable. A prodrug may be inactive whenadministered to a subject, but is converted in vivo to an activecompound. The use of prodrugs is particularly advantageous forfacilitating the administration of the compounds. The prodrug compoundoften offers benefits of solubility, tissue compatibility or delayedrelease in a mammalian organism. For example the prodrug, according tothe principles of the present invention, can be a compound representedby the structure of formula I wherein R¹, R², R⁵ and R⁶ are a functionalgroup that gives rise to hydroxyl upon hydrolysis as definedhereinabove.

All stereoisomers of the above compounds are contemplated, either inadmixture or in pure or substantially pure form. The compounds can haveasymmetric centers at any of the atoms. Consequently, the compounds canexist in enantiomeric or diastereomeric forms or in mixtures thereof.The present invention contemplates the use of any racemates (i.e.mixtures containing equal amounts of each enantiomers), enantiomericallyenriched mixtures (i.e., mixtures enriched for one enantiomer), pureenantiomers or diastereomers, or any mixtures thereof. The chiralcenters can be designated as R or S or R,S or d,D, l,L or d,l, D,L.Compounds comprising amino acid residues include residues of D-aminoacids, L-amino acids, or racemic derivatives of amino acids. Compoundscomprising sugar residues include residues of D-sugars, L-sugars, orracemic derivatives of sugars. Residues of D-sugars, which appear innature, are preferred. In addition, several of the compounds of theinvention contain one or more double bonds. The present inventionintends to encompass all structural and geometrical isomers includingcis, trans, E and Z isomers, independently at each occurrence.

One or more of the compounds of the invention, may be present as a salt.The term “salt” encompasses both basic and acid addition salts,including but not limited to, carboxylate salts or salts with aminenitrogens, and include salts formed with the organic and inorganicanions and cations discussed below. Furthermore, the term includes saltsthat form by standard acid-base reactions with basic groups (such asamino groups) and organic or inorganic acids. Such acids includehydrochloric, hydrofluoric, trifluoroacetic, sulfuric, phosphoric,acetic, succinic, citric, lactic, maleic, fumaric, palmitic, cholic,pamoic, mucic, D-glutamic, D-camphoric, glutaric, phthalic, tartaric,lauric, stearic, salicylic, methanesulfonic, benzenesulfonic, sorbic,picric, benzoic, cinnamic, and like acids. Each possibility represents aseparate embodiment of the invention.

The term “organic or inorganic cation” refers to counter-ions for theanion of a salt. The counter-ions include, but are not limited to,alkali and alkaline earth metals (such as lithium, sodium, potassium,barium, aluminum and calcium); ammonium and mono-, di- and tri-alkylamines such as trimethylamine, cyclohexylamine; and the organic cations,such as dibenzylammonium, benzylammonium, 2-hydroxyethylammonium,bis(2-hydroxyethyl)ammonium, phenylethylbenzylammonium,dibenzylethylenediammonium, and like cations. See, for example, Berge etal., J. Pharm. Sci. (1977), 66:1-19, which is incorporated herein byreference.

The present invention also includes solvates of the compounds of thepresent invention and salts thereof. “Solvate” means a physicalassociation of a compound of the invention with one or more solventmolecules. This physical association involves varying degrees of ionicand covalent bonding, including hydrogen bonding. In certain instancesthe solvate will be capable of isolation. “Solvate” encompasses bothsolution-phase and isolatable solvates. Non-limiting examples ofsuitable solvates include ethanolates, methanolates and the like.“Hydrate” is a solvate wherein the solvent molecule is water.

The present invention also includes polymorphs of the compounds of thepresent invention and salts thereof. The term “polymorph” refers to aparticular crystalline or amorphous state of a substance, which can becharacterized by particular physical properties such as X-raydiffraction, IR or Raman spectra, melting point, and the like.

IRS/Stat3 Dual Modulators and EGFR Inhibitors/Antibody Combinations

In one embodiment, the present invention relates to a method of treatinga tumor that has developed resistance to an Epidermal Growth FactorReceptor (EGFR) inhibitor and/or EGFR antibody, the method comprisingthe step of contacting the tumor with an EGFR inhibitor and/or EGFRantibody in combination with a compound of any of formulae (I), (II),(III), (IV), or any of the individual compounds covered by suchformulae.

In another embodiment, the present invention relates to a method ofpreventing acquired resistance of a tumor to an Epidermal Growth FactorReceptor (EGFR) inhibitor and/or EGFR antibody, the method comprisingthe step of contacting the tumor with an EGFR inhibitor and/or EGFRantibody in combination with a compound of any of formulae (I), (II),(III), (IV), or any of the individual compounds covered by suchformulae.

In another embodiment, the present invention relates to a pharmaceuticalcombination comprising a compound represented by the structure of any offormulae (I), (II), (III), (IV), or any of the individual compoundscovered by such formulae, in combination with an Epidermal Growth Factor(EGFR) inhibitor and/or EGFR antibody.

In other embodiments, the present invention further relates to thecombination as described above for use in treating a tumor that isresistant to an EGFR inhibitor and/or EGFR antibody, or for preventingacquired resistance to an EGFR inhibitor and/or EGFR antibody.

In other embodiments, the present invention further relates to the useof the combination described above for the preparation of a medicamentfor the treatment of a tumor that is resistant to an EGFR inhibitorand/or EGFR antibody, or for preventing acquired resistance to an EGFRinhibitor and/or EGFR antibody.

In some embodiments, the tumor is present in a cancer patient havingtumors with acquired resistance to EGFR inhibitor and/or EGFR antibodytreatment. In other embodiments, the treatment results in attenuation orregression in the growth of the resistant tumors. In other embodiments,the tumor is present in a cancer patient who is receiving treatment withan EGFR inhibitor and/or EGFR antibody or is a candidate for receivingsuch treatment.

Any EGFR inhibitor or antibody known to a person of skill in the art maybe used in the combinations of the present invention. In someembodiments, the EGFR inhibitor is selected from the group consisting oferlotinib, gefitinib, lapatinib, vandetanib, neratinib, icotinib,afatinib, dacomitinib, poziotinib, AZD9291, CO-1686, HM61713 andAP26113. In one currently preferred embodiment, the EGFR inhibitor iserlotinib. In one specific embodiment, the compound is represented bythe structure of formula D and the EGFR inhibitor is erlotinib. Eachpossibility represents a separate embodiment of the present invention.

In some embodiments, the EGFR antibody is selected from the groupconsisting of trastuzumab, necitumumab. cetuximab and panitumumab. Eachpossibility represents a separate embodiment of the present invention.

In one embodiment, the compound is a compound of formula A. In anotherembodiment, the compound is a compound of formula B. In anotherembodiment, the compound is a compound of formula C. In anotherembodiment, the compound is a compound of formula D. In anotherembodiment, the compound is a compound of formula E. In anotherembodiment, the compound is a compound of formula F. In anotherembodiment, the compound is a compound of formula G. In anotherembodiment, the compound is a compound of formula H. In anotherembodiment, the compound is a compound of formula I. In anotherembodiment, the compound is a compound of formula J. In anotherembodiment, the compound is a compound of formula IV-4. In one currentlypreferred embodiment, the compound is represented by the structure offormula D. However, it is apparent to one of skill in the art that anyof the compounds described herein may be used in the combinations of thepresent invention.

IRS/Stat3 Dual Modulators and mTOR Inhibitor Combinations

In further aspects of the present invention, it has now unexpectedlybeen found that a combination of an IRS/Stat3 dual modulator of any offormulae (I), (II), (III), (IV), or any of the individual compoundscovered by such formulae as described herein, and an inhibitor ofmammalian target of rapamycin (mTOR), provides a therapeutic effect thatis at least additive, and is preferably synergistic as compared with thetreatment effect of each agent alone. Furthermore, the combination canbe used to treat a tumor that has developed resistance to an mTORinhibitor, and/or to prevent acquired resistance of a tumor to the mTORinhibitor.

Accordingly, in one embodiment, the present invention relates to apharmaceutical combination comprising a compound represented by thestructure of any of formulae (I), (II), (III), (IV), or any of theindividual compounds covered by such formulae and at least one inhibitorof mammalian target of rapamycin (mTOR), wherein the compound and the atleast one mTOR inhibitor together provide a synergistic therapeuticanti-cancer effect

In another embodiment, the present invention relates to a method oftreating cancer, comprising the step of administering to the subject inneed thereof a therapeutically effective amount of a pharmaceuticalcombination comprising a compound represented by the structure of any offormulae (I), (II), (III), (IV), and at least one inhibitor of mammaliantarget of rapamycin (mTOR), wherein the compound and the at least onemTOR inhibitor together provide a synergistic therapeutic effect.

In another embodiment, the present invention relates to a method oftreating a tumor that has developed resistance to an inhibitor mammaliantarget of Rapamycin (mTOR), the method comprising the step of contactingthe tumor with mTOR inhibitor in combination with a Compound of any offormulae (I), (II), (III), (IV), or any of the individual compoundscovered by such formulae.

In another embodiment, the present invention relates to a method ofpreventing acquired resistance of a tumor to an inhibitor mammaliantarget of Rapamycin (mTOR), the method comprising the step of contactingthe tumor with an mTOR inhibitor in combination with a Compound of anyof formulae (I), (II), (III), (IV), or any of the individual compoundscovered by such formulae.

In other embodiments, the present invention further relates to thecombination as described above for use in treating a tumor that isresistant to an mTOR inhibitor, or for preventing acquired resistance toan mTOR inhibitor.

In other embodiments, the present invention further relates to the useof the combination described above for the preparation of a medicamentfor the treatment of a tumor that is resistant to an mTOR inhibitor, orfor preventing acquired resistance to an mTOR inhibitor.

In other embodiments, the present invention further relates to acombination as described above, for use in treating a tumor that isresistant to an mTOR inhibitor, or for preventing acquired resistance toan mTOR inhibitor.

In some embodiments, the tumor is present in a cancer patient havingtumors with acquired resistance to mTOR inhibitor treatment. In otherembodiments, the treatment results in attenuation or regression in thegrowth of the resistant tumors. In other embodiments, the tumor ispresent in a cancer patient who is receiving treatment with an mTORinhibitor or is a candidate for receiving such treatment.

Any mTOR inhibitor known to a person of skill in the art may be used inthe combinations of the present invention. In some embodiments, the mTORinhibitor is a first generation inhibitor selected from the groupconsisting of rapamycin (Sirolimus); Ridaforolimus (Deforolimus,AP23573, MK-8669); NVP-BEZ235(2-Methyl-2-{4-[3-methyl-2-oxo-8-(quinolin-3-yl)-2,3-dihydro-1H-imidazo[4,5-c]quinolin-1-yl]phenyl}propanenitrile);Everolimus (Afinitor, RAD-001, the 40-O-(2-hydroxyethyl) derivative ofsirolimus); and Temsirolimus (CCI-779). In a currently preferredembodiment, thee mTOR inhibitor is Everolimus.

In other embodiments, the mTOR inhibitor is a second generation compound(inhibitor of mTORC1 and mTORC2), such as OSI-027(trans-4[4-Amino-5-(7-methoxy-1H-indol-2-yl)imidazo[5,1-f][1,2,4]triazin-7-yl]cyclohexanecarboxylicacid); XL765 (SAR245409); INK128(3-(2-amino-5-benzoxazolyl)-1-(1-methylethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine);MLN0128, AZD2014(3-(2,4-bis((S)-3-methylmorpholino)pyrido[2,3-d]pyrimidin-7-yl)-N-methylbenzamide);DS-3078a and Palomid529(3-(4-methoxybenzyloxy)-8-(1-hydroxyethyl)-2-methoxy-6H-benzo[c]chromen-6-one).

In one specific embodiment, the compound is represented by the structureof formula D and the mTOR inhibitor is Everolimus (Afinitor).

In one embodiment, the compound is a compound of formula A. In anotherembodiment, the compound is a compound of formula B. In anotherembodiment, the compound is a compound of formula C. In anotherembodiment, the compound is a compound of formula D. In anotherembodiment, the compound is a compound of formula E. In anotherembodiment, the compound is a compound of formula F. In anotherembodiment, the compound is a compound of formula G. In anotherembodiment, the compound is a compound of formula H. In anotherembodiment, the compound is a compound of formula I. In anotherembodiment, the compound is a compound of formula J. In anotherembodiment, the compound is a compound of formula IV-4. In one currentlypreferred embodiment, the compound is represented by the structure offormula D. However, it is apparent to one of skill in the art that anyof the compounds described herein may be used in the combinations of thepresent invention.

IRS/Stat3 Dual Modulators and Immunotherapy Agent Combinations

In one embodiment, the present invention relates to a method ofsensitizing a tumor to immunotherapy, the method comprising the step ofcontacting the tumor with a compound of any of formulae (I), (II),(III), (IV), or any of the individual compounds covered by such formulaein combination with an immunotherapy agent.

In another embodiment, the present invention relates to a pharmaceuticalcombination comprising a compound represented by the structure of any offormulae (I), (II), (III), (IV), or any of the individual compoundscovered by such formulae, in combination with an immunotherapy agent.

In other embodiments, the present invention further relates to thecombination as described above for use in the treatment of a tumor bysensitizing the tumor to immunotherapy.

In other embodiments, the present invention further relates to the useof the combination described above for the preparation of a medicamentfor the treatment of a tumor by sensitizing the tumor to immunotherapy.

In some embodiments, the tumor is present in a cancer patient who isreceiving immunotherapy or is a candidate for receiving immunotherapy.

Any immunotherapy agent that is known to a person of skill in the artcan be used in the combination of the present invention. In someembodiments, the immunotherapy agent is an antibody against a targetselected from the group consisting of PDL, PD1, CTLA4, CD20, CD30, CD33,CD52, VEGF, CD30, EGFR and ErbB2. In some embodiments, the antibody isselected from the group consisting of Alemtuzumab (Campath®),Bevacizumab (Avastin®), Brentuximab vedotin (Adcetris®), Cetuximab(Erbitux®), Gemtuzumab ozogamicin (Mylotarg®), Ibritumomab tiuxetan(Zevalin®), Ipilimumab (Yervoy®), Ofatumumab (Arzerra®), Panitumumab(Vectibix®), Rituximab (Rituxan®), Tositumomab (Bexxar®) and Tratuzumab(Herceptin®). Each possibility represents a separate embodiment of thepresent invention.

In one embodiment, the compound is a compound of formula A. In anotherembodiment, the compound is a compound of formula B. In anotherembodiment, the compound is a compound of formula C. In anotherembodiment, the compound is a compound of formula D. In anotherembodiment, the compound is a compound of formula E. In anotherembodiment, the compound is a compound of formula F. In anotherembodiment, the compound is a compound of formula Ga. In anotherembodiment, the compound is a compound of formula H. In anotherembodiment, the compound is a compound of formula I. In anotherembodiment, the compound is a compound of formula J. In anotherembodiment, the compound is a compound of formula IV-4. In one currentlypreferred embodiment, the compound is represented by the structure offormula D. However, it is apparent to one of skill in the art that anyof the compounds described herein may be used in the combinations of thepresent invention.

IRS/Stat3 Dual Modulators and Mitogen-Activated Protein Kinase (MEK)Inhibitor and/or a Mutated B-Raf Inhibitor Combinations

In other aspects, it has now unexpectedly been found that a combinationof a dual modulator of Insulin Receptor Substrate (IRS) and signaltransducer and activator of transcription 3 (Stat3), as describedherein, and a mitogen-activated protein kinase (MEK) inhibitor and/or amutated B-Raf inhibitor, provides a therapeutic effect that is at leastadditive, and is preferably synergistic as compared with the treatmenteffect of each agent alone. Furthermore, the combination can be used totreat a tumor that has developed resistance to a MEK inhibitor and/ormutated B-Raf inhibitor, and/or to prevent acquired resistance of atumor to a MEK inhibitor and/or mutated B-Raf inhibitor and/or toprevent or delay delaying tumor recurrence following cease of treatmentwith a MEK inhibitor and/or mutated B-Raf inhibitor.

Thus, in some embodiments, the present invention relates to a method oftreating a tumor that has developed resistance to a mitogen-activatedprotein kinase (MEK) inhibitor and/or a mutated B-Raf inhibitor, themethod comprising the step of contacting the tumor with a MEK inhibitorand/or mutated B-Raf inhibitor, in combination with a compoundrepresented by the structure of formula (III) or (IV).

In other embodiments, the present invention relates to method ofpreventing acquired resistance of a tumor to a MEK inhibitor and/ormutated B-Raf inhibitor, the method comprising the step of contactingthe tumor with a MEK inhibitor and/or mutated B-Raf inhibitor, incombination with a compound represented by the structure of formula(III) or (IV).

In other embodiments, the present invention relates to a method ofpreventing or delaying tumor recurrence following cease of treatmentwith a MEK inhibitor and/or a mutated B-Raf inhibitor, the methodcomprising the step of contacting the tumor with a MEK inhibitor and/ormutated B-Raf inhibitor, in combination with a compound represented bythe structure of formula (III) or (IV).

In other embodiments, the present invention relates to a pharmaceuticalcombination comprising a compound represented by the structure offormula (III), in combination with a mitogen-activated protein kinase(MEK) inhibitor, and optionally a mutated B-Raf inhibitor. In someembodiments, the combination comprises a compound of formula (III), aMEK inhibitor and a mutated B-Raf inhibitor preferably, wherein the MEKinhibitor is Trametinib, and the mutated B-Raf inhibitor is Vemurafenib.

In other embodiments, the present invention relates to a compoundrepresented by the structure of formula (IV), in combination with amitogen-activated protein kinase (MEK) inhibitor, and/or a mutated B-Rafinhibitor.

In some embodiments, the tumor is present in a cancer patient havingtumors with acquired resistance to MEK inhibitor and/or mutated B-Rafinhibitor treatment. In other embodiments, the treatment results inattenuation or regression in the growth of the resistant tumors. Inother embodiments, the tumor is present in a cancer patient who isreceiving treatment with an MEK inhibitor and/or a mutated B-Rafinhibitor or is a candidate for receiving such treatment.

Any MEK inhibitor known to a person of skill in the art may be used inthe combinations of the present invention. In some embodiments, the MEKinhibitor is selected from the group consisting of Trametinib(GSK1120212), Selumetinib, Binimetinib (MEK162), PD-325901, Cobimetinib,CI-1040 and PD035901, preferably, wherein the MEK inhibitor is Trametin.

Any mutated B-Raf inhibitor known to a person of skill in the art may beused in the combinations of the present invention. In some embodiments,the mutated B-Raf inhibitor is selected from the group consisting ofVemurafenib (PLX-4032), PLX4720, Sorafenib (BAY43-9006), and Dabrafenib,preferably, wherein the mutated B-Raf inhibitor is Vemurafenib.

In one embodiment, the compound is represented by the structure offormula (III). In another embodiment, the compound is represented by thestructure of formula (IV). Each possibility represents a separateembodiment of the present invention.

In some embodiments, the compound is represented by the structure offormula D and the MEK inhibitor is Trametinib.

In other embodiments, the compound is represented by the structure offormula D and the mutated B-Raf inhibitor is Vemurafenib.

In some embodiments, the combination treatment includes a compound offormula (III) or (IV), and either a MEK inhibitor or a mutated B-Rafinhibitor. In other embodiments, the combination treatment includes acompound of formula (III) or (IV), and both a MEK inhibitor and amutated B-Raf inhibitor.

In some embodiments, the present invention relates to a pharmaceuticalcombination comprising a compound represented by the structure offormula D in combination with Trametinib.

In other embodiments, the present invention relates to a pharmaceuticalcombination comprising a compound represented by the structure offormula D in combination with Trametinib and Vemurafenib.

In other embodiments, the present invention relates to the combinationsdescribed above, for use in treating a tumor that is resistant to a MEKinhibitor and/or a mutated B-Raf inhibitor, or for preventing acquiredresistance to a MEK inhibitor and/or a mutated B-Raf inhibitor.

In other embodiments, the present invention relates to the use of thecombinations described above, for the preparation of a medicament forthe treatment of a tumor that is resistant to a MEK inhibitor and/or amutated B-Raf inhibitor, or for preventing acquired resistance to a MEKinhibitor and/or a mutated B-Raf inhibitor.

In one embodiment, the compound is a compound of formula A. In anotherembodiment, the compound is a compound of formula B. In anotherembodiment, the compound is a compound of formula C. In anotherembodiment, the compound is a compound of formula D. In anotherembodiment, the compound is a compound of formula E. In anotherembodiment, the compound is a compound of formula F. In anotherembodiment, the compound is a compound of formula Ga. In anotherembodiment, the compound is a compound of formula H. In anotherembodiment, the compound is a compound of formula I. In anotherembodiment, the compound is a compound of formula J. In anotherembodiment, the compound is a compound of formula IV-4. In one currentlypreferred embodiment, the compound is represented by the structure offormula D. However, it is apparent to one of skill in the art that anyof the compounds described herein may be used in the combinations of thepresent invention.

IRS/Stat3 Dual Modulators and Chemotherapeutic Agent Combinations

In other aspects, it has now unexpectedly been found that a combinationof a dual modulator of Insulin Receptor Substrate (IRS) and signaltransducer and activator of transcription 3 (Stat3), as describedherein, and a chemotherapeutic agent such as Gemcitabine, 5-FU,Irinotecan, Oxaliplatin and any combination thereof (e.g., thecombination treatment FOLFIRI or FOLFOX), provides a therapeutic effectthat is at least additive, and is preferably synergistic as comparedwith the treatment effect of each agent alone. Furthermore, thecombination can be used to treat a tumor that has developed resistanceto any of these chemotherapeutic agents or their combination and/or toprevent acquired resistance of a tumor to any of these chemotherapeuticagents or their combination, and/or to prevent or delay delaying tumorrecurrence following cease of treatment with any of these therapeuticagents or their combination.

FOLFIRI is a combination treatment for cancer containing Leucovorin(Folinic Acid), 5-FU and Irinotecan. FOLFOX is a combination treatmentfor cancer containing Leucovorin calcium (Folinic Acid), 5-FU andOxaliplatin.

Thus, according to some embodiments, the present invention relates to apharmaceutical combination comprising a compound represented by thestructure of formula (III) or (IV) and at least one chemotherapeuticagent selected from Gemcitabine, 5-FU, Irinotecan, Oxaliplatin and anycombination thereof, wherein the compound and the chemotherapeuticagent(s) together provide a synergistic therapeutic anti-cancer effect.

In some embodiments, the present invention relates to a method oftreating cancer, comprising the step of administering to the subject inneed thereof a therapeutically effective amount of a combinationcomprising a compound represented by the structure of formula (III) or(IV) and at least one chemotherapeutic agent selected from Gemcitabine,5-FU, Irinotecan, Oxaliplatin and any combination thereof, wherein thecompound and the chemotherapeutic agent(s) together provide asynergistic therapeutic anti-cancer effect.

In other embodiments, the present invention provides a method oftreating a tumor that has developed resistance to at least onechemotherapeutic agent, e.g., Gemcitabine, 5-FU, Irinotecan, Oxaliplatinand any combination thereof, the method comprising the step ofcontacting the tumor with at least one of said chemotherapeutic agent(s)in combination with a compound represented by the structure of formula(III) or (IV).

In other embodiments, the present invention provides a method ofpreventing acquired resistance of a tumor to at least onechemotherapeutic agent, e.g., Gemcitabine, 5-FU, Irinotecan, Oxaliplatinand any combination thereof, the method comprising the step ofcontacting the tumor with at least one of said chemotherapeutic agent(s)in combination with a compound represented by the structure of formula(III) or (IV).

In other embodiments, the present invention provides a method ofpreventing or delaying tumor recurrence following cease of treatmentwith at least one chemotherapeutic agent, e.g., Gemcitabine, 5-FU,Irinotecan, Oxaliplatin and any combination thereof, the methodcomprising the step of contacting the tumor with at least one of saidchemotherapeutic agent(s) in combination with a compound represented bythe structure of formula (III) or (IV).

In some embodiments, the tumor is present in a cancer patient havingtumors with acquired resistance to said chemotherapeutic agent(s). Inother embodiments, the treatment results in attenuation or regression inthe growth of the resistant tumors. In other embodiments, the tumor ispresent in a cancer patient who is receiving treatment with saidchemotherapeutic agent(s), or is a candidate for receiving suchtreatment.

In other embodiments, the present invention provides a pharmaceuticalcombination comprising a compound represented by the structure offormula (III) or (IV) and at least one chemotherapeutic agent, e.g.,Gemcitabine, 5-FU, Irinotecan, Oxaliplatin and any combination thereof,for use in treating a tumor that is resistant to said chemotherapeuticagent(s), or for preventing acquired resistance to said chemotherapeuticagent(s), or for delaying tumor recurrence following cease of treatmentwith such chemotherapeutic agent(s).

In other embodiments, the present invention relates to the use of apharmaceutical combination comprising a compound represented by thestructure of formula (III) or (IV) and at least one chemotherapeuticagent, e.g., Gemcitabine, 5-FU, Irinotecan, Oxaliplatin and anycombination thereof, for the preparation of a medicament for thetreatment of a tumor that is resistant to said chemotherapeuticagent(s), or for preventing acquired resistance to said chemotherapeuticagent(s), or for of preventing or delaying tumor recurrence followingcease of treatment with such chemotherapeutic agent(s).

It is apparent to a person of skill in the art that otherchemotherapeutic agents related to the above non-limiting examples canbe used in the combinations of the present invention. For example, thepresent invention contemplates the use of other platinum compounds(e.g., carboplatin and cisplatin), SN-38 (a metabolite of Irinotecan)and other fluoropyrimidines (analogs of 5-FU).

In one embodiment, the compound is a compound of formula A. In anotherembodiment, the compound is a compound of formula B. In anotherembodiment, the compound is a compound of formula C. In anotherembodiment, the compound is a compound of formula D. In anotherembodiment, the compound is a compound of formula E. In anotherembodiment, the compound is a compound of formula F. In anotherembodiment, the compound is a compound of formula Ga. In anotherembodiment, the compound is a compound of formula H. In anotherembodiment, the compound is a compound of formula I. In anotherembodiment, the compound is a compound of formula J. In anotherembodiment, the compound is a compound of formula IV-4. In one currentlypreferred embodiment, the compound is represented by the structure offormula D. However, it is apparent to one of skill in the art that anyof the compounds described herein may be used in the combinations of thepresent invention.

Treatment of Cancer

The term “cancer” as used herein refers to a disorder in which apopulation of cells has become, in varying degrees, unresponsive to thecontrol mechanisms that normally govern proliferation anddifferentiation. Cancer refers to various types of malignant neoplasmsand tumors, including primary tumors, and tumor metastasis. Non-limitingexamples of cancers which can be treated by the combinations of thepresent invention are brain, ovarian, colon, prostate, kidney, bladder,breast, lung, oral, and skin cancers. Specific examples of cancers are:carcinomas, sarcomas, myelomas, leukemias, lymphomas and mixed typetumors. Particular categories of tumors include lymphoproliferativedisorders, breast cancer, ovarian cancer, prostate cancer, cervicalcancer, endometrial cancer, bone cancer, liver cancer, stomach cancer,colon cancer, pancreatic cancer, cancer of the thyroid, head and neckcancer, cancer of the central nervous system, cancer of the peripheralnervous system, skin cancer, kidney cancer, as well as metastases of allthe above. Particular types of tumors include hepatocellular carcinoma,hepatoma, hepatoblastoma, rhabdomyosarcoma, esophageal carcinoma,thyroid carcinoma, ganglioblastoma, fibrosarcoma, myxosarcoma,liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, Ewing's tumor, leimyosarcoma, rhabdotheliosarcoma,invasive ductal carcinoma, papillary adenocarcinoma, melanoma, squamouscell carcinoma, basal cell carcinoma, adenocarcinoma (welldifferentiated, moderately differentiated, poorly differentiated orundifferentiated), renal cell carcinoma, hypernephroma, hypernephroidadenocarcinoma, bile duct carcinoma, choriocarcinoma, seminoma,embryonal carcinoma, Wilms' tumor, testicular tumor, lung carcinomaincluding small cell, non-small and large cell lung carcinoma, bladdercarcinoma, glioma, astrocyoma, medulloblastoma, craniopharyngioma,ependymoma, pinealoma, retinoblastoma, neuroblastoma, colon carcinoma,rectal carcinoma, hematopoietic malignancies including all types ofleukemia and lymphoma including: acute myelogenous leukemia, acutemyelocytic leukemia, acute lymphocytic leukemia, chronic myelogenousleukemia, chronic lymphocytic leukemia, mast cell leukemia, multiplemyeloma, myeloid lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma,and hepatocarcinoma. Each possibility represents a separate embodimentof the present invention.

In some representative embodiments, the cancer is selected from thegroup consisting of head and neck (H&N) cancer, sarcoma, multiplemyeloma, ovarian cancer, breast cancer, kidney cancer, stomach cancer,hematopoietic cancers, lymphoma, leukemia, including lymphoblasticleukemia, lung carcinoma, melanoma, glioblastoma, hepatocarcinoma,prostate cancer and colon cancer. Each possibility represents a separateembodiment of the present invention.

The term “treatment of cancer” in the context of the present inventionincludes at least one of the following: a decrease in the rate of growthof the cancer (i.e. the cancer still grows but at a slower rate);cessation of growth of the cancerous growth, i.e., stasis of the tumorgrowth, and, in preferred cases, the tumor diminishes or is reduced insize. The term also includes reduction in the number of metastases,reduction in the number of new metastases formed, slowing of theprogression of cancer from one stage to the other and a decrease in theangiogenesis induced by the cancer. In most preferred cases, the tumoris totally eliminated. Additionally included in this term is lengtheningof the survival period of the subject undergoing treatment, lengtheningthe time of diseases progression, tumor regression, and the like. It isto be understood that the term “treating cancer” also refers to theinhibition of a malignant (cancer) cell proliferation including tumorformation, primary tumors, tumor progression or tumor metastasis. Theterm “inhibition of proliferation” in relation to cancer cells, mayfurther refer to a decrease in at least one of the following: number ofcells (due to cell death which may be necrotic, apoptotic or any othertype of cell death or combinations thereof) as compared to control;decrease in growth rates of cells, i.e. the total number of cells mayincrease but at a lower level or at a lower rate than the increase incontrol; decrease in the invasiveness of cells (as determined forexample by soft agar assay) as compared to control even if their totalnumber has not changed; progression from a less differentiated cell typeto a more differentiated cell type; a deceleration in the neoplastictransformation; or alternatively the slowing of the progression of thecancer cells from one stage to the next.

As used herein, the term “administering” refers to bringing in contactwith the combination of the present invention. Administration can beaccomplished to cells or tissue cultures, or to living organisms, forexample humans. In one embodiment, the present invention encompassesadministering the combinations of the present invention to a humansubject.

A “therapeutic” treatment is a treatment administered to a subject whoexhibits signs of pathology for the purpose of diminishing oreliminating those signs. A “therapeutically effective amount” is thatamount of compound or a composition which is sufficient to provide abeneficial effect to the subject to which the compound or composition isadministered.

The term “following cease of treatment” as used herein means aftertreatment with the drug of choice is stopped. For example, according tocertain embodiments of the present invention, the IRS/Stat3 DualModulator (e.g., compound of formula (III) or (IV)) is administeredtogether (sequentially or concurrently) with any of the combinationtreatments described herein, for a desired duration of time. Then,treatment (with all compounds) is stopped and the tumors are monitoredfor a desired period of time. As contemplated herein, the IRS/Stat3 DualModulators of the present invention are able to prevent or delay tumorrecurrence following cease of treatment with the any of the combinationdrugs described herein, to a greater extent than any of these drugsadministered alone.

The term “treating a tumor that has developed resistance” to a certainanti-cancer drug, or “preventing acquired resistance of a tumor” to acertain anti-cancer drug, means any one or more of the following: (i)the tumors acquire or develop resistance as a result of treatment tothat anti-cancer drug; (ii) that the tumors acquire or developresistance as a result of treatment with other anti-cancer drugs; or(iii) the tumors have a primary resistance to that anti-cancer drug.

The combination therapy can provide a therapeutic advantage in view ofthe differential toxicity associated with the two individual treatments.For example, treatment with one compound can lead to a particulartoxicity that is not seen with the other compound, and vice versa. Assuch, this differential toxicity can permit each treatment to beadministered at a dose at which the toxicities do not exist or areminimal, such that together the combination therapy provides atherapeutic dose while avoiding the toxicities of each of theconstituents of the combination agents. Furthermore, when thetherapeutic effects achieved as a result of the combination treatmentare enhanced or synergistic, i.e., significantly better than additivetherapeutic effects, the doses of each of the agents can be reduced evenfurther, thus lowering the associated toxicities to an even greaterextent.

The terms “synergistic”, “cooperative” and “super-additive” and theirvarious grammatical variations are used interchangeably herein. Aninteraction between an IRS/Stat3 dual modulator and another anti-canceragent (e.g., mTOR inhibitor, EGFR inhibitor, EGFR antibody and/orimmunotherapy agent) is considered to be synergistic, cooperative orsuper-additive when the observed effect (e.g., cytotoxicity) in thepresence of the drugs together is higher than the sum of the individualeffects of each drug administered separately. In one embodiment, theobserved combined effect of the drugs is significantly higher than thesum of the individual effects. The term significant means that theobserved p<0.05. A non-limiting manner of calculating the effectivenessof the combined treatment comprises the use of the Bliss additivismmodel (Cardone et al. Science (1998), 282: 1318-1321) using thefollowing formula: Ebliss=EA+EB−EA×EB, where EA and EB are thefractional inhibitions obtained by drug A alone and drug B alone atspecific concentrations. When the experimentally measured fractionalinhibition is equal to Ebliss, the combination provides an additivetherapeutic effect. When the experimentally measured fractionalinhibition is greater than Ebliss, the combination provides asynergistic therapeutic effect.

Pharmaceutical Compositions

Although the components of the combinations of the present invention canbe administered alone, it is contemplated that the components areadministered in pharmaceutical compositions further containing at leastone pharmaceutically acceptable carrier or excipient. Each of thecomponents can be administered in a separate pharmaceutical composition,or the combination can be administered in one pharmaceuticalcomposition.

The pharmaceutical compositions of the present invention can beformulated for administration by a variety of routes including oral,rectal, transdermal, parenteral (subcutaneous, intraperitoneal,intravenous, intra-arterial, transdermal and intramuscular), topical,intranasal, or via a suppository. Each possibility represents a separateembodiment of the present invention. Such compositions are prepared in amanner well known in the pharmaceutical art and comprise as an activeingredient at least one compound of the present invention as describedhereinabove, and a pharmaceutically acceptable excipient or a carrier.The term “pharmaceutically acceptable” means approved by a regulatoryagency of the Federal or a state government or listed in the U.S.Pharmacopeia or other generally recognized pharmacopeia for use inanimals and, more particularly, in humans.

During the preparation of the pharmaceutical compositions according tothe present invention the active ingredient is usually mixed with acarrier or excipient, which may be a solid, semi-solid, or liquidmaterial. The compositions can be in the form of tablets, pills,capsules, pellets, granules, powders, lozenges, sachets, cachets,elixirs, suspensions, dispersions, emulsions, solutions, syrups,aerosols (as a solid or in a liquid medium), ointments containing, forexample, up to 10% by weight of the active compound, soft and hardgelatin capsules, suppositories, sterile injectable solutions, andsterile packaged powders. Each possibility represents a separateembodiment of the present invention.

The carriers may be any of those conventionally used and are limitedonly by chemical-physical considerations, such as solubility and lack ofreactivity with the compound of the invention, and by the route ofadministration. The choice of carrier will be determined by theparticular method used to administer the pharmaceutical composition.Some examples of suitable carriers include lactose, glucose, dextrose,sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate,alginates, tragacanth, gelatin, calcium silicate, microcrystallinecellulose, polyvinylpyrrolidone, cellulose, water and methylcellulose.Each possibility represents a separate embodiment of the presentinvention. The formulations can additionally include lubricating agentssuch as talc, magnesium stearate, and mineral oil; wetting agents,surfactants, emulsifying and suspending agents; preserving agents suchas methyl- and propylhydroxybenzoates; sweetening agents; flavoringagents, colorants, buffering agents (e.g., acetates, citrates orphosphates), disintegrating agents, moistening agents, anti-bacterialagents, anti-oxidants (e.g., ascorbic acid or sodium bisulfite),chelating agents (e.g., ethylenediaminetetraacetic acid), and agents forthe adjustment of tonicity such as sodium chloride. Other pharmaceuticalcarriers can be sterile liquids, such as water and oils, including thoseof petroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like, polyethylene glycols,glycerin, propylene glycol or other synthetic solvents. Water is apreferred carrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. Each possibility represents a separate embodimentof the present invention.

For preparing solid compositions such as tablets, the principal activeingredient(s) is mixed with a pharmaceutical excipient to form a solidpreformulation composition containing a homogeneous mixture of acompound of the present invention. When referring to thesepreformulation compositions as homogeneous, it is meant that the activeingredient is dispersed evenly throughout the composition so that thecomposition may be readily subdivided into equally effective unit dosageforms such as tablets, pills and capsules. This solid preformulation isthen subdivided into unit dosage forms of the type described abovecontaining, for example, from about 0.1 mg to about 2000 mg, from about0.1 mg to about 500 mg, from about 1 mg to about 100 mg, from about 100mg to about 250 mg, etc. of the active ingredient(s) of the presentinvention.

Any method can be used to prepare the pharmaceutical compositions. Soliddosage forms can be prepared by wet granulation, dry granulation, directcompression and the like. The solid dosage forms of the presentinvention may be coated or otherwise compounded to provide a dosage formaffording the advantage of prolonged action. For example, the tablet orpill can comprise an inner dosage and an outer dosage component, thelatter being in the form of an envelope over the former. The twocomponents can be separated by an enteric layer, which serves to resistdisintegration in the stomach and permit the inner component to passintact into the duodenum or to be delayed in release. A variety ofmaterials can be used for such enteric layers or coatings, suchmaterials including a number of polymeric acids and mixtures ofpolymeric acids with such materials as shellac, cetyl alcohol, andcellulose acetate. Each possibility represents a separate embodiment ofthe present invention.

The liquid forms in which the compositions of the present invention maybe incorporated, for administration orally or by injection, includeaqueous solutions, suitably flavored syrups, aqueous or oil suspensions,and flavored emulsions with edible oils such as cottonseed oil, sesameoil, coconut oil, or peanut oil, as well as elixirs and similarpharmaceutical vehicles. Each possibility represents a separateembodiment of the present invention.

Compositions for inhalation or insulation include solutions andsuspensions in pharmaceutically acceptable aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions maycontain suitable pharmaceutically acceptable excipients as describedabove. In one embodiment, the compositions are administered by the oralor nasal respiratory route for local or systemic effect. Compositions inpharmaceutically acceptable solvents may be nebulized by use of inertgases. Nebulized solutions may be breathed directly from the nebulizingdevice or the nebulizing device may be attached to a face masks tent, orintermittent positive pressure breathing machine. Solution, suspension,or powder compositions may be administered, orally or nasally, fromdevices that deliver the formulation in an appropriate manner

Another formulation suitable for the compositions and methods of thepresent invention employs transdermal delivery devices (“patches”). Suchtransdermal patches may be used to provide continuous or discontinuousinfusion of the compounds of the present invention in controlledamounts. The construction and use of transdermal patches for thedelivery of pharmaceutical agents is well known in the art.

In yet another embodiment, the composition is prepared for topicaladministration, e.g. as an ointment, a gel a drop or a cream. Fortopical administration to body surfaces using, for example, creams,gels, drops, ointments and the like, the compounds of the presentinvention can be prepared and applied in a physiologically acceptablediluent with or without a pharmaceutical carrier. The present inventionmay be used topically or transdermally to treat cancer, for example,melanoma. Adjuvants for topical or gel base forms may include, forexample, sodium carboxymethylcellulose, polyacrylates,polyoxyethylene-polyoxypropylene-block polymers, polyethylene glycol andwood wax alcohols. Each possibility represents a separate embodiment ofthe present invention.

Alternative formulations include nasal sprays, liposomal formulations,slow-release formulations, pumps delivering the drugs into the body(including mechanical or osmotic pumps) controlled-release formulationsand the like, as are known in the art.

The compositions are preferably formulated in a unit dosage form. Theterm “unit dosage forms” refers to physically discrete units suitable asunitary dosages for human subjects and other mammals, each unitcontaining a predetermined quantity of active material(s) calculated toproduce the desired therapeutic effect, in association with a suitablepharmaceutical excipient.

In preparing a formulation, it may be necessary to mill the activeingredient to provide the appropriate particle size prior to combiningwith the other ingredients. If the active compound is substantiallyinsoluble, it ordinarily is milled to a particle size of less than 200mesh. If the active ingredient is substantially water soluble, theparticle size is normally adjusted by milling to provide a substantiallyuniform distribution in the formulation, e.g. about 40 mesh.

It may be desirable to administer the pharmaceutical composition of theinvention locally to the area in need of treatment; this may be achievedby, for example, and not by way of limitation, local infusion duringsurgery, infusion to the liver via feeding blood vessels with or withoutsurgery, topical application, e.g., in conjunction with a wound dressingafter surgery, by injection, by means of a catheter, by means of asuppository, or by means of an implant, the implant being of a porous,non-porous, or gelatinous material. According to some embodiments,administration can be by direct injection e.g., via a syringe, at thesite of a tumor or neoplastic or pre-neoplastic tissue.

The compounds may also be administered by any convenient route, forexample by infusion or bolus injection, by absorption through epitheliallinings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and maybe administered together with other therapeutically active agents. Theadministration may be localized or it may be systemic. In addition, itmay be desirable to introduce the pharmaceutical compositions of theinvention into the central nervous system by any suitable route,including intra-ventricular and intrathecal injection; intra-ventricularinjection may be facilitated by an intra-ventricular catheter, forexample, attached to a reservoir. Pulmonary administration can also beemployed, e.g., by use of an inhaler or nebulizer, and formulation withan aerosolizing agent.

A compound of the present invention can be delivered in an immediaterelease or in a controlled release system. In one embodiment, aninfusion pump may be used to administer a compound of the invention,such as one that is used for delivering chemotherapy to specific organsor tumors (see Buchwald et al., 1980, Surgery 88: 507; Saudek et al.,1989, N. Engl. J. Med. 321: 574). In one embodiment, a compound of theinvention is administered in combination with a biodegradable,biocompatible polymeric implant, which releases the compound over acontrolled period of time at a selected site. Examples of polymericmaterials include, but are not limited to, polyanhydrides,polyorthoesters, polyglycolic acid, polylactic acid, polyethylene vinylacetate, copolymers and blends thereof. In yet another embodiment, acontrolled release system can be placed in proximity of the therapeutictarget, thus requiring only a fraction of the systemic dose.

Furthermore, at times, the pharmaceutical compositions may be formulatedfor parenteral administration (subcutaneous, intravenous,intra-arterial, transdermal, intraperitoneal or intramuscular injection)and may include aqueous and non-aqueous, isotonic sterile injectionsolutions, which can contain anti-oxidants, buffers, bacteriostats, andsolutes that render the formulation isotonic with the blood of theintended recipient, and aqueous and non-aqueous sterile suspensions thatinclude suspending agents, solubilizers, thickening agents, stabilizers,and preservatives. Oils such as petroleum, animal, vegetable, orsynthetic oils and soaps such as fatty alkali metal, ammonium, andtriethanolamine salts, and suitable detergents may also be used forparenteral administration. The above formulations may also be used fordirect intra-tumoral injection. Further, in order to minimize oreliminate irritation at the site of injection, the compositions maycontain one or more nonionic surfactants. Suitable surfactants includepolyethylene sorbitan fatty acid esters, such as sorbitan monooleate andthe high molecular weight adducts of ethylene oxide with a hydrophobicbase, formed by the condensation of propylene oxide with propyleneglycol.

The parenteral formulations can be presented in unit-dose or multi-dosesealed containers, such as ampoules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example, water, for injections, immediatelyprior to use. Extemporaneous injection solutions and suspensions can beprepared from sterile powders, granules, and tablets of the kindpreviously described and known in the art. Each possibility represents aseparate embodiment of the present invention.

Alternatively, the combinations of the present invention can be used inhemodialysis such as leukophoresis and other related methods, e.g.,blood is drawn from the patient by a variety of methods such as dialysisthrough a column/hollow fiber membrane, cartridge etc., is treated withthe IRS/Stat3 dual modulator and/or additional anti-cancer agentex-vivo, and returned to the patient following treatment. Such treatmentmethods are well known and described in the art. See, e.g., Kolho et al.(J. Med. Virol. 1993, 40(4):318-21); Ting et al. (Transplantation, 1978,25(1):31-3); the contents of which are hereby incorporated by referencein their entirety.

Doses and Dosing Schedules

The treatment with the IRS/Stat3 dual modulator and the otheranti-cancer agent (i.e., EGFR inhibitor/EGFR antibody/mTORinhibitor/immunotherapy agent/MEK inhibitor/mutated B-Rafinhibitor/chemotherapeutic agent or combination thereof) can take placesequentially in any order, simultaneously or a combination thereof. Forexample, administration of an IRS/Stat3 dual modulator can take placeprior to, after or at the same time as administration of the otheranti-cancer agent or combination thereof. For example, a total treatmentperiod can be decided for the IRS/Stat3 dual modulator. The otheranti-cancer agent can be administered prior to onset of treatment withthe IRS/Stat3 dual modulator or following treatment with the IRS/Stat3dual modulator. In addition, the other anti-cancer agent can beadministered during the period of IRS/Stat3 dual modulatoradministration but does not need to occur over the entire treatmentperiod. In another embodiment, the treatment regimen includespre-treatment with one agent, either the IRS/Stat3 dual modulator or theEGFR inhibitor/EGFR antibody/mTOR inhibitor/immunotherapy agent/MEKinhibitor/mutated B-Raf inhibitor/chemotherapeutic agent or combinationthereof followed by the addition of the other agent or agents.Alternating sequences of administration are also contemplated.Alternating administration includes administration of an IRS/Stat3 dualmodulator and other anti-cancer agent in alternating sequences, e.g.,IRS/Stat3 dual modulator, followed by the other anti-cancer agent,followed by IRS/Stat3 dual modulator, etc.

The amount of a compound that will be effective in the treatment of aparticular disorder or condition, including cancer, will depend on thenature of the disorder or condition, and can be determined by standardclinical techniques. In addition, in vitro assays may optionally beemployed to help identify optimal dosage ranges. The precise dose to beemployed in the formulation will also depend on the route ofadministration, and the progression of the disease or disorder, andshould be decided according to the judgment of the practitioner and eachpatient's circumstances. A preferred dosage will be within the range of0.01-1000 mg/kg of body weight, 0.1 mg/kg to 100 mg/kg, 1 mg/kg to 100mg/kg, 10 mg/kg to 75 mg/kg, 0.1-1 mg/kg, etc. Exemplary (non-limiting)amounts of the IRS/Stat3 dual modulator EGFR inhibitor/EGFRantibody/mTOR inhibitor/immunotherapy agent/MEK inhibitor/mutated B-Rafinhibitor/chemotherapeutic agent include 0.1 mg/kg, 0.2 mg/kg, 0.5mg/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, 50 mg/kg, 60 mg/kg, 75mg/kg and 100 mg/kg. Alternatively, the amount administered can bemeasured and expressed as molarity of the administered compound. By wayof illustration and not limitation, an IRS/Stat3 dual modulator (e.g. acompound of any of formulae I, II, III, IV) can be administered in arange of 0.1-10 mM, e.g., 0.1, 0.25, 0.5, 1 and 2 mM. Alternatively, theamount administered can be measured and expressed as mg/ml, μg/ml, orng/ml. By way of illustration and not limitation, the EGFRinhibitor/EGFR antibody/mTOR inhibitor/immunotherapy agent/MEKinhibitor/mutated B-Raf inhibitor/chemotherapeutic agent can beadministered in an amount of 1 ng/ml to 100 mg/ml, for example 1-1000ng/ml, 1-100 ng/ml, 1-1000 μg/ml, 1-100 μg/ml, 1-1000 mg/ml, 1-100mg/ml, etc. Effective doses may be extrapolated from dose-responsecurves derived from in vitro or animal model test bioassays or systems.When a synergistic effect is observed, the overall dose of each of thecomponents may be lower, thus the side effects experienced by thesubject may be significantly lower, while a sufficient anti-cancereffect is nevertheless achieved.

In one embodiment, the combination therapy reduces the amount of each ofits component by a factor of 2, i.e., each component is given at halfthe dose as compared with single agent therapy, and still achieves thesame or similar therapeutic effect. In another embodiment, thecombination therapy reduces the amount of each of its component by afactor of 5, 10, 20, 50 or 100. As demonstrated herein, the IC₅₀ ofchemotherapeutic agents as anti-proliferative agents in various cancercells are reduced as compared to the IC₅₀ of the chemotherapeutic agent,when administered alone.

The administration schedule will depend on several factors such as thecancer being treated, the severity and progression, the patientpopulation, age, weight etc. For example, the compositions of theinvention can be taken once-daily, twice-daily, thrice daily,once-weekly or once-monthly. In addition, the administration can becontinuous, i.e., every day, or intermittently. The terms “intermittent”or “intermittently” as used herein means stopping and starting at eitherregular or irregular intervals. For example, intermittent administrationcan be administration one to six days per week or it may meanadministration in cycles (e.g. daily administration for two to eightconsecutive weeks, then a rest period with no administration for up toone week) or it may mean administration on alternate days. The differentcomponents of the combination can, independently of the other, followdifferent dosing schedules.

The following examples are presented in order to more fully illustratecertain embodiments of the invention. They should in no way, however, beconstrued as limiting the broad scope of the invention. One skilled inthe art can readily devise many variations and modifications of theprinciples disclosed herein without departing from the scope of theinvention.

EXPERIMENTAL DETAILS SECTION Example 1: Prevention of AcquiredResistance to Erlotinib with Compound D

Experimental system: Patient-derived xenograft (PDX) of Squamous Cellcarcinoma of Head&Neck (SCCHN) tumor biopsy subcutaneous implanted intoNodScid mice.

I. Animals and Biopsy

-   -   Biopsy: Fresh human primary SCCHN tumor biopsy.

Tumor Type: Salivary gland mucoepidermoid carcinoma.

Genomic analysis revealed amplified and mutated (activated) EGFR.

-   -   Implantation of tumor biopsy grafts (P0): Fresh human primary        SCCHN tumor biopsy grafts were sub-cutaneously (SC) implanted        into 5 female NOD.CB17-Prkdc^(scid)/J (NodScid mice), 5-6 weeks        old (Harlan, IL), following 14 days of acclimation.    -   Implantation of tumor biopsy grafts (P1) into NodScid mice for        efficacy study: 3.5 weeks following implantation of the biopsy        (P0), tumors reached average size of about 1,200 mm³, the mice        were sacrificed by cervical dislocation and the tumors were        excised. The tumors were measured, cut into small pieces of 2-4        mm and transferred into a gentleMACS Tube containing sterile        saline. Tumor volume was adjusted with saline to get 1.5 mm³        tumor volume/100 μl saline. The sample was dissociated using a        gentleMACS Octo Dissociator. The dissociated tumor tissue was        collected with 18G syringe and injected directly under the skin.        35 female NodScid mice 4-5 weeks old (Harlan, IL) were injected        each subcutaneously into the nape area with 100 μl of the        obtained cell solution (approximately 1.5 mm³ tumor volume P1 in        100 μl saline per mouse) Animals were observed and monitored for        any discomfort and immobility day by day, checked for inability        to move or feed properly, being hunched and inactive, and        ulcerations, defined as exposing of necrotic centers.    -   Onset of tumor growth (palpable tumor mass) was detected ten        days following cell injection. After 8 days, 32 out of 35        injected mice developed tumors with an average size of about 80        mm³. The mice were randomly divided into 4 treatment groups        including 8 animals/group.

II. Treatments and Procedures

When tumor size was ˜80 mm³ (day0) the following treatments initiated:

-   -   1. Control (vehicle): Water 100 μl PO (5 times/week, daily)    -   2. Compound D 70 mg/kg in 20% 2-Hydroxypropyl-β-cyclodextrin        (HPbCD), IV (3 times/week, qod)    -   3. Erlotinib 100 mg/kg PO (5 times/week, daily).    -   4. Erlotinib 100 mg/kg PO (5 times/week)+Compound D 70 mg/kg IV        (3 times/week). Erlotinib was administered ˜4 hr following        Compound D, when administered on the same days.

All treatments for each of the treatment groups 1-4 were initiatedsimultaneously.

The length (l) and the width (w) of the tumors were measured 4 times aweek and the volumes of the tumors were calculated as follows: v=lw²/2.Graphs represent average tumor volumes with standard errors (standarddeviations/square root of group size). Mice weight and behavior wereexamined at least once a week. After two weeks of treatment mice weresacrificed and the tumors were excised for biochemical and genomicanalysis 14 hrs following last administration of drug/inhibitor. Threemice in the combined treatment group were not sacrificed at the end oftreatment and were kept with no further treatment.

Results

As shown in FIG. 1, treatment with Erlotinib, an EGFR TK inhibitor,initially led to a significant tumor regression in all the treated mice(FIG. 1, open squares). However, after one week of treatment all thetumors developed resistance to Erlotinib and progressed aggressively.Combined treatment with Erlotinib and Compound D led to significanttumor regression in all the treated mice and none of the tumors regrewduring the period of combined treatment (FIG. 1, open circles).

Two mice that achieved complete response were kept alive with no furthertreatment and remained free of disease after 3 months with no furthertreatment.

Although the initial tumor has not responded to Compound D alone, theacquired resistance to Erlotinib was completely abolished by Compound D.Evidence from the literature suggests that treatment with Erlotinibinduces IRS up-regulation leading to resistance by the activation ofIGF1R/IRS-to-AKT survival pathway. Other reports claim that Stat3phosphorylation is induced by Erlotinib in H&N cancer, and theinhibition of Stat3 & EGFR has synergic inhibitory effect on H&N tumors.Without wishing to be bound by any particular theory or mechanism ofaction, Compound D and other compounds of formulae (I-IV) describedherein are dual inhibitors of IRS1/2 and Stat3 and, therefore, shouldantagonize these Erlotinib-induced mechanisms and prevent resistance.

Example 2: Regression of Erlotinib-Resistant Tumors with CombinedTreatment of Erlotinib and Compound D

Experimental system: Patient-derived xenograft (PDX) of Squamous Cellcarcinoma of Head&Neck (SCCHN) tumor biopsy subcutaneous implanted intoNodScid mice.

I. Animals and Biopsy

-   -   Implantation of SCCHN tumor biopsy graft (P8) into NodScid mice        for efficacy study: Five months following implantation of SCCHN        tumor biopsy graft (P1) described above, tumor cells (P8) were        injected into NodScid mice from self-breeding, 9.5 weeks old,        using the same procedure described for implantation of P1. The        original biopsy is the same as described above and the P        indicates passages (implantation number in mice).    -   Onset of tumor growth (palpable tumor mass) was detected seven        days following cell injection. 12 days later, mice which        developed tumors sized around 70 mm³. The mice were randomly        divided into 4 treatment groups including 4 animals in the        groups treated with Vehicle, Compound D or Compound D+        Erlotinib, and the rest treated with Erlotinib. Treatments        initiated simultaneously (day 0).

II. Treatments and Procedures

Treatment groups included:

-   -   1. Vehicle-control: 20% 2-Hydroxypropyl-β-cyclodextrin (HPbCD)        50 μl/injection, IV (3 times/week, qod).    -   2. Compound D 70 mg/kg in HPbCD, IV (3 times/week, qod).    -   3. Erlotinib 100 mg/kg in HPbCD, PO (5 times/week).    -   4. Erlotinib 100 mg/kg PO (5 times/week)+Compound D 70 mg/kg IV        (3 times/week). Erlotinib was administered ˜4 hr following        Compound D, when administered at the same days.

All these treatments were initiated simultaneously.

Treatment with Erlotinib (Group 3) led to a dramatic tumor regression(FIG. 2, open squares). While on treatment, tumors developed resistanceto Erlotinib after 1 week of treatment and aggressively progressed.Erlotinib-treated mice, which developed tumors around 130 mm³ on day10(n=7), were split to two groups:

-   -   5. The first (n=3) continued to get Erlotinib (100 mg/kg PO, 5        times/week), and    -   6. The second group (n=4) started a combined treatment with        Erlotinib (100 mg/kg PO, 5 times/week)+Compound D (70 mg/kg IV,        3 times/week, qod) on day10 of treatment. Erlotinib was        administered ˜4 hr following Compound D, when administered at        the same days.

The length (l) and the width (w) of the tumors were measured 4 times aweek and the volumes of the tumors were calculated as follows: v=lw²/2.Graphs represent average tumor volumes with standard errors (standarddeviations/square root of group size). Mice weight and behavior wereexamined at least once a week. Mice were sacrificed and the tumors wereexcised for biochemical and genomic analysis.

Results

As shown in FIG. 2, treatment with Erlotinib (open square) led to tumorregression in 78% of treated mice (14 out of 18 mice responded). Howeverwhile on treatment, tumors developed resistance to Erlotinib after 1week and aggressively progressed. Erlotinib-treated mice whose tumorswere ˜130 mm³ on day 10 (n=7) were split to two groups—the first (n=3)continued to get Erlotinib (open squares), and the second group (n=4)started a combined treatment with Erlotinib+Compound D (black circles)on day10 of treatment. Dramatic tumor regression was observed followinginitiation of the combined treatment (FIG. 2, late treatment, blackcircles) while tumors of mice treated with Erlotinib only, aggressivelydeveloped (FIG. 2, open squares). Combined treatment withErlotinib+Compound D initiated on day 0 (FIG. 2, early treatment, opencircles) led to a significant tumor regression in all treated mice andno tumors regrew, consistent with the results of Example 1.

Conclusion

In conclusion, combined treatment of Compound D+Erlotinib is highlyeffective and leads to a dramatic regression of tumors followingresistance to Erlotinib has already acquired. Furthermore, in earlytreatment of established tumors Compound D prevents acquired resistanceto Erlotinib.

Example 3: Compound D Prevents Acquired Resistance to Erlotinib Evenwhen Initial Tumor Size is Very High (700 mm³)

Experimental system: Patient-derived xenograft (PDX) of Squamous Cellcarcinoma of SCCHN tumor biopsy subcutaneous implanted into NRG mice.

I. Animals and Biopsy

-   -   Implantation of SCCHN tumor biopsy graft (P11) into NRG mice for        efficacy study: Eight months following implantation of SCCHN        tumor biopsy graft (P1) described above, tumor cells (P11) were        injected into 20 male mice NOD.Cg-Rag1tm1Mom Il2rgtm1Wj1/SzJ        mice (Common name: NRG), from self-breeding, using the same        procedure described for implantation of P1. The original biopsy        is the same as described above and the P indicates passages        (implantation number in mice).    -   Onset of tumor growth (palpable tumor mass) was detected six        days following cell injection. 13 days later, 19 out of 20        injected mice developed tumors with an average size of 700-720        mm³ (day0). The mice were randomly divided into 4 treatment        groups including 4 animals in the group treated with Vehicle,        and 5 mice/group in the groups treated with Erlotinib, Compound        D or Compound D+Erlotinib. All treatments initiated        simultaneously on day 0.

II. Treatments and Procedures

Treatment groups included:

-   -   1. Vehicle-control: 20% HPbCD 50 μl/injection, IV (3 times/week,        qod), 4 mice.    -   2. Compound D 70 mg/kg in HPbCD, IV (3 times/week, qod), 5 mice.    -   3. Erlotinib 100 mg/kg in HPbCD, PO (5 times/week), 5 mice.    -   4. Erlotinib 100 mg/kg PO (5 times/week)+Compound D 70 mg/kg IV        (3 times/week), 5 mice. Erlotinib was administered ˜4 hr        following Compound D, when administered at the same days.

The length (l) and the width (w) of the tumors were measured 4 times aweek and the volumes of the tumors were calculated as follows: v=lw²/2.Graphs represent average tumor volumes with standard errors (standarddeviations/square root of group size). Mice weight and behavior wereexamined routinely. Mice were sacrificed and the tumors were excised forbiochemical and genomic analysis.

Results

As shown in FIG. 3, treatment with Erlotinib led to a significantresponse of the tumors, their growth was halted and they regressed.However while on treatment, tumors developed resistance to Erlotinib aweek following treatment initiation and aggressively progressed (FIG. 3,open squares). Combined treatment with Erlotinib+Compound D initiated onday 0 (FIG. 3, open circles) showed the same response as to Erlotinib inthe first week but the combined treatment with Compound D preventedacquired resistance to Erlotinib, prevented regrowth of tumors andinduced tumor regression, consistent with the results of Example 1.

Conclusion

In conclusion, combined treatment of Compound D+Erlotinib is highlyeffective and prevents acquired resistance to Erlotinib, even if theinitial size of the tumors was very high (700 mm³) when treatmentsinitiated.

Example 4: Combined Treatment of Compound D and Afinitor EfficientlyBlocks the Growth of Sarcoma Patient-Derived Xenografts in Mice

Experimental system: Patient-derived xenograft (PDX) of UteralAdenoSarcoma biopsy subcutaneous implanted into NodScid mice.

I. Animals and Biopsy

-   -   Biopsy: Frozen human primary Uteral AdenoSarcoma (sample ID:        OT_001)    -   Implantation of tumor biopsy grafts (P0): Frozen human primary        Uteral AdenoSarcoma biopsy grafts were sub-cutaneous (SC)        implanted (P0) into female NOD.CB17-Prkdc^(scid)/J (NodScid        mice, Harlan IL). Three months later the tumors were excised,        cut into small pieces and implanted in 38 NodScid mice (P1) for        efficacy study.    -   Eight days following implantation of the biopsy (P1) tumor onset        was detected in 37 mice.    -   A week later (day0), tumors in 33 mice reached an average size        of 130 mm³ and the mice were randomly divided into 4 treatment        groups.

II. Treatments and Procedures

Treatment groups included:

-   -   1. Control: 20% HPbCD 50 ul IP, qod (6 mice).    -   2. Compound D 70 mg/kg in 20% HPbCD, IV, qod (6 mice).    -   3. Afinitor 5 mg/kg PO, qod (15 mice).    -   4. Afinitor 5 mg/kg PO (qod)+Compound D 70 mg/kg IV (qod), 6        mice. Afinitor was administered ˜4 hr following Compound D.

All treatments were initiated simultaneously on day0.

The length (l) and the width (w) of the tumors were measured every otherday and the volumes of the tumors were calculated as follows: v=lw²/2.Graphs represent average tumor volumes with standard errors (standarddeviations/square root of group size). Four days following initiation oftreatment the tumors of the control group and the Compound D groupalready reached the end point and mice were sacrificed.

Results

As shown in FIG. 4, treatment with Afinitor (open squares), an mTOR/S6Kinhibitor, led to growth inhibition of the tumors: while average tumorsize in the control group increased 16-fold the average tumor size inthe Afinitor group increased 5.5-fold.

Surprisingly, although Compound D alone (open triangles) had nosignificant effect on tumor growth, the combined treatment ofAfinitor+Compound D (open circles) led to tumor regression. The averagetumor size of the combined treatment regressed from 130 mm³ to 70 mm³ infour days and after only two treatments.

In terms of responders vs. non-responder mice, while no response toCompound D alone was detected, and only half of the mice in theAfinitor-treated group responded (group A, n=8) and half—not (group B,n=7), all the mice in the combined treatment responded and most tumorseven significantly regressed.

Example 5: Compound D Prevents Acquired Resistance to Afinitor (A) andLeads to Regression of Afinitor-Resistant Tumors (B)

Experimental system: Patient-derived xenograft (PDX) of UteralAdenoSarcoma biopsy subcutaneous implanted into NodScid mice, describedin example 4.

The experiment described in Example 4 (phase I) was extended to phase II(FIG. 5A) and phase III (FIG. 5B) of the experiment. Followingtreatments described in phase I (example 4), the mice whose tumorsreached the end point were sacrificed, and the following treatments werecontinued.

Phase II:

-   -   1. The Afinitor-responder group (group A, open squares) was        administered with Afinitor 5 mg/kg PO, qd (8 mice).    -   2. The combined treatment group (open circles) continued to get        the Afinitor 5 mg/kg PO (qod)+Compound D 70 mg/kg IV (qod)        treatment (6 mice). Afinitor was administered ˜4 hr following        Compound D.

Phase III:

The tumors in the Afinitor-responder group (group A) regressed, butwhile on treatment acquired resistance to Afinitor and aggressivelyprogressed to average tumor size of 590 mm³ on day6. The group wasdivided to two groups of 4 mice each which received the followingtreatments on day6 onwards:

-   -   The first continued to get Afinitor 5 mg/kg PO, qd (4 mice)    -   and the second got Afinitor 5 mg/kg PO (qod)+Compound D 70 mg/kg        IV (qod) combined treatment (4 mice). Afinitor was administered        ˜4 hr following Compound D.

The length (l) and the width (w) of the tumors were measured every otherday and the volumes of the tumors were calculated as follows: v=lw²/2.The graph in FIG. 5A represents average tumor volumes with standarderrors (standard deviations/square root of group size). The graph inFIG. 5B represents growth rates in %, while the 100% for each tumor wasdefined as its volume at day 6.

Results

The Afinitor-treated group was split to responders (open squares, groupA, n=8) vs. non-responders (grey squares, group B, n=7). Afinitortreatment of group A initially induced tumor regression (tumorsregressed from average tumor size of 125 mm³ on day0 to 37 mm³ on day5),but while on treatment all tumors developed resistance to Afinitor andaggressively progressed (to average tumor size of 590 mm³ on day6).

Combined treatment of Afinitor and Compound D from day0 (treatmentinitiation) induced tumor regression and their average tumor volumeremained low till the end of the experiment (FIG. 5A, ∘). Although theinitial tumor has not responded to Compound D alone, the acquiredresistance to Afinitor was completely abolished by Compound D. Evidencefrom the literature suggests that treatment with Afinitor induces IRSup-regulation leading to resistance by the activation ofIGF1R/IRS-to-AKT survival pathway. mTOR/S6K is a negative regulator ofthe IRS proteins. It phosphorylates IRS on Serine residues and therebydown-regulates its levels and decreases its affinity to receptortyrosine kinases (RTK) IGF1R and IR. Inhibition of mTOR/S6K shouldstabilize IRS1/2, increase their levels and enhance their complexationwith IGF1R and IR, leading to the activation of AKT survival pathway andacquired resistance to mTOR inhibitors. This feedback loop was describedin the literature (Crose L. E. S. and Linardic C. M. Sarcoma 2011,Keniry M. and Parsons R. Cancer Discovery 2011; 1:203-204) and it hasbeen shown that AKT phosphorylation is a clinically observablephenomenon following treatment with the mTOR inhibitorAfinitor/Everolimus in women with breast cancer. Without wishing to bebound by any particular theory or mechanism of action, it is believedthat that eliminating IRS1/2 from the cancer cell by IRS/Stat3 dualmodulators such as Compound D and other compounds of formulae (I-IV)described herein will prevent acquired resistance to Afinitor or anyother mTOR inhibitor, and may synergize with these inhibitors afterresistance has already acquired to induce tumor regression.

Following resistance to Afinitor has been acquired, the mice of group Awere divided into two groups, the first remained on Afinitor alone (□)and the second received combined treatment of Afinitor+Compound Dstarting on day 6 of treatment (●). While tumors significantlyprogressed under treatment with Afinitor alone (□), the combinedtreatment of Compound D and Afinitor induced tumor regression (●). Thegraph in FIG. 5B represents growth rates in %, while the 100% for eachtumor was defined as its volume at day 6.

Example 5A: Combined Treatment with Afinitor+Compound D of HighlyAggressive Cancer with No Available Medical Treatment Delayed AcquiredResistance to Afinitor and Achieved Complete Response in 40% of theGroup

Experimental system: Patient-derived xenograft (PDX) of UteralAdenoSarcoma biopsy subcutaneous implanted into NodScid mice, describedin example 4.

The experiment described in Example 4 was repeated in purchased micefrom Harlan.

Treatments:

-   -   1. Control: 20% HPbCD 50 ul IP, qod (3 mice).    -   2. Compound D 70 mg/kg in 20% HPbCD, IV, 3 time a week, qod (3        mice).    -   3. Afinitor 5 mg/kg PO, 4 times a week (17 mice).    -   4. Afinitor 5 mg/kg PO (qod)+Compound D 70 mg/kg IV (qod), 3        time a week (5 mice). Afinitor was administered ˜4 hr following        Compound D.    -   Treatments ceased on day17.

The length (l) and the width (w) of the tumors were measured every otherday and the volumes of the tumors were calculated as follows: v=lw²/2.Graphs represent average tumor volumes with standard errors (standarddeviations/square root of group size). Five days following initiation oftreatment the tumors of the control group and the Compound D groupalready reached the end point and mice were sacrificed.

Results

As shown in previous experiment, although Compound D alone (opentriangles) had no effect on tumor growth, the combined treatment ofAfinitor+Compound D (black circles) led to tumor regression. The tumorsin the Afinitor-responder group (14 out of 17 treated mice) regressed,but while on treatment, after one week of treatment, acquired resistanceto Afinitor and aggressively progressed (open squares). Combinedtreatment of Afinitor and Compound D significantly delayed acquiredresistance to Afinitor in 60% of the group (3 out of 5 treated mice,open cycles dashed line) and completely erased the tumors in 40% of thegroup (2 out of 5 treated mice, open cycles continuous line). These twomice were kept alive with no further treatment and remained free ofdisease after more than 3 months with no further treatment (FIG. 5C).

Example 6 I. Cell Lines

-   -   A375 (human melanoma), HCT15 (colon cancer), SK-ES.1 (Ewing        sarcoma), NCI-H460 (lung cancer) and PC3 (prostate cancer) were        cultured in RPMI with 10% fetal calf serum (FCS).    -   HepG2 (hepatocarcinoma) were cultured in Dulbecco's Modified        Eagle Medium (DMEM) and F12 (1:1) containing 10% FCS    -   DU145 (prostate cancer) were cultured in RPMI containing 5% FCS        and 5 mg/L insulin.

All cell lines were obtained from the American Type Culture Collection.YUMAC, YURIF, YUSIK (all human melanoma, kindly provided by Prof. RuthHalaban, Yale University, New Haven, Conn.) were cultured in OptiMEMcontaining 5% FCS. M571, M2068, M560n (all human melanoma, kindlyprovided by Dr. Michal Lotem, Hadassah Hospital, Jerusalem, Israel) weremaintained in RPMI, DMEM and F12 (1:3:1) containing 10% FCS. 451-Lu(human melanoma) and 451-Lu-BR (PLX4032-resistant melanoma; ref. 32)were maintained in RPMI containing 5% FCS (media for resistant linescontained 1 mmol/L PLX4032). All media were supplemented with 100 U/mLpenicillin and 100 mg/mL streptomycin and all cells were grown at 37°C./5% CO₂.

All melanoma cells used and discussed in FIGS. 6-9 and table 1 are fromhuman origin and bearing the mutated BRAF^(600K/E).

II. Cell Proliferation

Cells were grown in complete medium and treated with inhibitors one dayfollowing seeding. 72 hours later, the surviving cells were quantifiedby methylene blue staining or by WST-1 staining for nonadherent cells(Roche).

III. Immunoblots

-   -   Cells were treated as indicated in FIGS. 6-9 and the        corresponding figure legends, following overnight        serum-starvation (unless indicated differently). When cells were        treated with both PLX4032 and compounds A or D, PLX4032 was        added 3-4 hr following the compounds.    -   Cells were lysed with boiling sample buffer (10% glycerol, 50        mmol/L Tris-HCl, pH 6.8, 3% SDS, and 5% 2-mercaptoethanol).        Western blot analysis was conducted in 8% SDS-PAGE, using        antibodies described below.    -   Aliquots of cell extracts containing equal amounts of protein        were resolved by 8% SDS/PAGE and electroblotted onto        nitrocellulose filters. The membranes were blocked with low-fat        milk diluted 1:20 in TBST (NaCl/Tris containing 0.2% Tween-20)        for 0.5 hr, incubated with Rabbit anti-phosphoY705-Stat3        antibody (Cell signaling cat #9131), Mouse        anti-ERK-diphosphorylated-YT (Sigma Aldrich cat #M8159) or        anti-PARP antibodies overnight at 4 C in 5% BSA in TBST        containing 0.05% azid, washed extensively with TBST and then        incubated with horseradish peroxidase-conjugated secondary        antibodies for 45 min at room temperature in 5% BSA in TB ST.    -   Immunoreactive bands were visualized using enhanced        chemiluminescence. Membranes were re-blotted with Mouse        anti-Stat3 antibody (Transduction labs cat #21320) or with        Rabbit anti AKT1/2 (Santa cruz cat #sc-8312) or Anti-Actin as        described above.

IV. Chemotaxis of Peripheral Blood Mononuclear Cells (PBMCs)

A375 cells were seeded in 96-well plates (6000 cells/well) and grownovernight. Cells were treated with Compound A and washed twice with themedium 4 hrs after treatment where indicated (Wash). 30 hrs followingtreatment 150 ul of medium were transferred to lower plate of chemotaxisdevice. 10,000 PBMCs/75 ul medium/well were added to the upper plate. Inaddition, PBMCs were added into lower plate as positive control (FIG.9B—Cell Titer Glo calibration graph, 10-10,000 cells/well). Chemotaxiswas examined 24 hrs later by Cell Titer Glo analysis of the lower plate.In addition, survival of A375 cells was analyzed by Methylene blue 30hrs after treatment with Compound A.

TABLE 1 The dual modulators of IRS/Stat3 potently inhibit theproliferation and viability of various cancer cells as compared to IGF1Rinhibitor OSI-906. IC50 (uM) Indication Cell line Compound D Compound AOSI-906 Prostate cancer PC3 0.5 0.8 >10 Melanoma Mel1617-Pa 0.2 0.3   >3Mel1617-BR 0.3 0.3   >3 451Lu-Pa 0.3 ND >>3 451Lu-BR 0.6 ~0.7 >>3 Coloncancer HCT15 0.8 ND >>3 Multipie Myeloma MM1S 0.2 0.3     0.2Hepatocarcinoma HepG2 0.7 1     8.3

Cells were plated in 96-well plates in 5-10% FCS in medium; a day laterexposed to various concentrations of compound A, compound D or OSI-906and 3 days later stained with methylene-blue and the relative cellnumber was quantified.

Results

Compounds A and D which were previously shown to induce IRS1/2 serinephosphorylation, were found to efficiently induce a reduction inY705-phosphorylation levels of Stat3 in cancer cells. These dualmodulators of IRS/Stat3 potently inhibit Stat3 phosphorylation (pStat3)in a dose-dependent manner (FIG. 6A) without affecting Stat3 proteinlevels. The inhibitory effect demonstrated by compound A and D ispotentiated with time: IC50 values of both compounds were ˜2 μM 1.3 hrpost-treatment and decreased to <1 μM 3 hr later. The describedinhibitory effect on Stat3 phosphorylation levels is long-term (FIG. 6B)as it can be detected long after the modulators were washed out thecells (FIG. 6C). FIG. 6C also demonstrates that a short exposure of A375melanoma cells to Compound D was sufficient to induce cell apoptosis 24and 48 hr later. The blockage of Stat3 Y705-phosphorylation isexemplified for compounds A, B, C, D, F, IV-1, IV-2, IV-3 and IV-4.

Since Stat3 is reported to be involved both in survival and drugresistance and in immune evasion of various cancer types, the ability ofthe Stat3/IRS dual modulators to sensitize tumors to specific PKinhibitory drugs and immunotherapies, respectively, was tested.

The phosphorylation levels of Stat3 in melanoma that acquired resistanceto BRAF inhibitor (BRAFi) such as PLX4032 (also known as Vemurafenib orZelboraf) is significantly higher as compared to the parent melanomacells/tumors (FIGS. 7A and 7B).This is shown in a metastatic melanomaclone 451-LU-BR [Villanueva et al. Cancer Cell 2010; 18:683-95] isolatedfollowing 6-months treatment with BRAFi (R), compared to the parentmetastatic melanoma 451-LU cell line before treatment (P). Furtherdemonstrated are the higher levels of Stat3 phosphorylation in cells (R)taken from patients (M2068, M560n, M571) that have been treated withPLX4032 and developed resistance towards it, compared to melanoma cellsfrom naïve patients (N) that carry mutated BRAF (YUMAC, YURIF, YUSIK)but were not yet treated with BRAFi (FIG. 7B).

The protein levels of Stat3 are similar in all samples, only thephosphorylation levels are dramatically enhanced in thePLX4032-resistant cells.

Surprisingly, it was discovered in PLX4032-sensitive melanoma cells thattreatment with 1 μM PLX4032 for 18-24 hr induced a marked induction inStat3 Y705-phosphorylation (pStat3). It was tested and demonstrated inthree different human metastatic melanoma cell lines (FIG. 7C-E). Theresults in FIG. 7 suggest that the increase in pStat3 may play a role inacquired resistance to BRAFi and that resistant cells adapt highconstant pStat3 level as a survival factor. Thereby it was speculatedthat combining dual IRS/Stat3 modulators with BRAFi may prevent acquiredresistance to BRAFi as well as to other drugs inducing up-regulation ofpStat3 and/or IRS1 and/or IRS2. The potential of the IRS/Stat3 dualmodulators to prevent acquired resistance to such drugs is indeeddemonstrated in Example 1, showing that compound D prevented acquiredresistance to Erlotinib in HNSCC derived from a patient and implanted tomice.

Furthermore, pStat3 has a major role in immune evasion of the tumor, itupregulates the expression and secretion of immune-suppressive factorsand down-regulates proinflammatory mediators, thereby masking the tumorsfrom the local immune system. In addition to cancer cells, diverseimmune subsets in the tumor micro-environment also displayconstitutively activated Stat3, and blocking Stat3 in immune cells mayalso elicit potent anti tumor immune response (increased cytotoxicity ofNK cells and neutrophils, T cell activation and increased tumorinfiltration, etc.). Therefore, it was speculated that combining ourdual IRS/Stat3 modulators with immunotherapy will down-regulate pSTAT3and sensitize the tumor to various immunotherapy agents.

Herein it is demonstrated that the IRS/Stat3 dual modulators,represented by compounds A & D, block both the basal and thePLX4032-induced levels of pStat3, while the IGF1R/IR TK inhibitorOSI-906 had no effect on pStat3 levels (FIGS. 7E & 8A). Testing thesignificance of this finding in terms of anti-cancer activity, theanti-proliferative activity of the IRS/Stat3 dual modulators vs. theIGF1R/IR TK inhibitor OSI-906 was compared in various cancer types.Table 1 shows that compounds A and D are far more effective than OSI-906in various melanoma cells (both PLX4032-resistant andPLX4032-sensitive); in colon cancer cells resistant to variouschemotherapies and EGFRi; in prostate cancer cells (resistant to severalchemotherapies) and hepatocellular carcinoma (resistant to EGFRi). Thesedifferences between the dual modulators and the tyrosine kinaseinhibitor of IGF1R/IR suggest that inhibiting both the Stat3 and IRS,central junction proteins highly involved in survival and drugresistance, may contribute to the potential of the dual modulators tosensitize resistant cancer cells to various therapies.

As previously described in FIGS. 6A&B, there are increased levels ofpStat3 in melanoma cells which developed resistance to BRAFi. FIGS. 8A&Bshow that compounds A & D block Stat3 phosphorylation completely inthese PLX4032-resistant clones of melanoma cell lines (451-LU-BRdescribed above and Mel1617-BR [Villanueva et al. Cancer Cell 2010;18:683-95]) as well as in melanoma cells derived from two patients[M2068 (i) & M571 (ii)] who have acquired resistance to PLX4032treatment (FIG. 8C). FIG. 8C demonstrates better activity of compound Dcompared with compound A. These results suggest that the IRS/Stat3 dualmodulators may re-sensitize melanoma cells which have acquiredresistance to BRAFi, and combined therapy of the IRS/Stat3 dualmodulators with BRAFi may induce tumor regression of the resistanttumors. The potential of the IRS/Stat3 dual modulators to re-sensitizedrug-resistant tumors to the drug, is indeed demonstrated in examples 2and 3, demonstrating that the combination of compound D with Erlotinibinduces regression of Erlotinib-resistant HNSCC tumors in mice.

The capability of the IRS/Stat3 dual modulators to inhibit pStat3 wasdemonstrated in various cancer types, as previously demonstrated fortheir effect on IRS1/2 Ser-phosphorylation and elimination. FIG. 8Dexemplifies their inhibitory activity on Stat3 Y705 phosphorylation(pStat3) in prostate cancer, multiple myeloma, Ewing sarcoma,hepatocellular carcinoma and NSCL.

The immune system is a powerful, largely untapped force for fightingtumors. Tumors have developed sophisticated mechanisms to evade theimmune system. Stat3 has a crucial role in mediating the crosstalkbetween tumor cells and tumor-interacting immune cells. Targeting Stat3in tumors involves bystander tumor cell killing associated withinfiltration of various immune effector cells. Thus, it was testedwhether treatment of tumor cells with IRS/Stat3 dual modulators mayinduce the recruitment of peripheral blood mononuclear cells towards thecancer cells. Human melanoma A375 cells were treated with increasingconcentrations of compound A and washed twice with the medium 4 hrspost-treatment where indicated (Wash). 30 hrs following treatment thecell medium was transferred to lower plate of chemotaxis device, and10,000 human PBMCs/well were added to the upper plate. Chemotaxis of thePBMCs towards the A375 medium samples was examined 24 hrs later by CellTiter Glo analysis of the lower plate As shown in FIG. 9A dose dependentchemotaxis was detected, suggesting compound A-regulated cytokineexpression/secretion inducing PBMC's recruitment towards the treatedtumor. Thus, combining our dual modulators with immunotherapy may gainenhanced anti-tumor effects. These IRS/Stat3 dual modulators shouldsensitize the tumors to other immunotherapies or PK inhibitors (EGFRi,mTORi etc) by affecting directly and indirectly the tumor cells and thetumor's microenvironment, including the tumor-interacting immune cells.

Example 7: Combined Treatment of EGFR Antibody Cetuximab with Compound DShows a Dramatic Delay in Tumor Recurrence Compared to Cetuximab Alonein Mice Implanted with a Tumor from a Head & Neck Squamous CellCarcinoma (HNSCC) Patient. The Same is True when Cetuximab+Afatinib areUsed Instead of Cetuximab

Experimental system: Patient-derived xenograft (PDX) of HNSCC tumorbiopsy subcutaneous implanted into NodScid mice.

I. Animals and Biopsy

-   -   Implantation of HNSCC tumor biopsy graft (P6) into NodScid mice        for efficacy study: Few months following implantation of frozen        HNSCC tumor biopsy graft (P1) in mice, tumor cells (P6) were        injected into NodScid mice (generated by in-house breeding),        using the same procedure described for implantation of P1. The        original biopsy is the same as described above and the P        indicates passages (implantation number in mice).    -   Onset of tumor growth (palpable tumor mass) was detected four        days following cell injection. Five days later, treatments        initiated in mice which developed tumors sized around 113 mm³.        The mice were randomly divided into 6 treatment groups including        4 animals in the groups treated with Cetuximab,        Cetuximab+Afatinib, Cetuximab+Compound D,        Cetuximab+Afatinib+Compound D, and 3 mice in the groups treated        with Vehicle or Compound D. Treatments initiated simultaneously        (on day 0) and applied for a period of 9 days.

II. Treatments and Procedures

Treatment groups included:

-   -   1. Vehicle-control: Vehicle (0.5% Hydroxymethyl-cellulose, 0.4%        Tween-80) 200 μl PO (5 times/week, qd).    -   2. Compound D 70 mg/kg in HPbCD, IV (3 times/week, qod).    -   3. Cetuximab 1 mg/mouse IP (2 times/week).    -   4. Cetuximab 1 mg/mouse IP (2 times/week)+Compound D 70 mg/kg IV        (3 times/week). Cetuximab was administered ˜4 hr following        Compound D, when administered at the same days.    -   5. Cetuximab 1 mg/mouse IP (2 times/week)+Afatinib 25 mg/kg in        vehicle PO (5 times/week)    -   6. Cetuximab 1 mg/mouse IP (2 times/week)+Afatinib 25 mg/kg PO        (5 times/week)+Compound D 70 mg/kg IV (3 times/week). Cetuximab        and/or Afatinib were administered ˜4 hr following Compound D,        when administered at the same days.

The length (l) and the width (w) of the tumors were measured 2-4 times aweek and the volumes of the tumors were calculated as follows: v=lw²/2.Graphs represent average tumor volumes with standard errors (standarddeviations/square root of group size). Mice weight and behavior wereexamined at least once a week. Mice were sacrificed and the tumors wereexcised for biochemical and genomic analysis.

Results

As shown in FIG. 10, at the first 4 days of treatment all tumorsprogressed, but then, treatment with either Cetuximab,Cetuximab+Compound D, Cetuximab+Afatinib or Cetuximab+Afatinib+CompoundD led to dramatic tumor regression in all mice, while all tumors in thevehicle-treated mice and in the Compound D-treated mice (as astand-alone treatment) aggressively progressed. Treatments applied for aperiod of 9 days only. Eight days following cease of treatments thetumors of the Cetuximab-treated group started re-growing andaggressively progressed and a week later the tumors of theCetuximab+Afatinib treated group progressed, while the combinations withCompound D extended the positive responses to >4 weeks following end oftreatment.

Afatinib is a second-generation irreversible EGFR tyrosine kinaseinhibitor developed to overcome acquired resistance to EGFR blockers,stems from EGFR T790M mutation, which is the most frequent mechanism ofacquired resistance to EGFR tyrosine kinase inhibitors.

Conclusion

Combined treatment of Compound D with either Cetuximab or evenCetuximab+Afatinib for 9 days only, significantly delayed recurrence ofregressed tumors and prolonged response to Cetuximab or toCetuximab+Afatinib.

Example 8: Compound D Synergizes with the Combination of Drugs,Comprising Inhibitors of Mutated-BRAF and MEK, to Induce Dramatic TumorRegression in Mice Implanted with Tumor Cells from a Melanoma PatientWho has Acquired Resistance to Vemurafenib

Experimental system: Patient-derived xenograft (PDX) of melanomasubcutaneous injected into NodScid mice.

I. Animals and Biopsy

-   -   Biopsy: Biopsy was excised from a melanoma patient harboring        mutated-BRaf^(V600E) who has shown short response to        Vemurafenib, and cells were seeded in plates. Early passage        cells (million cells/mouse) were sub-cutaneously (SC) injected        into NOD.CB17-Prkdc^(scid)/J (NodScid) female mice 10 weeks old        (generated by in-house breeding). Onset of tumors was detected        five days later, and treatments initiated seven days following        cell-injection when average tumor volume was ˜60 mm³.

II. Treatments and Procedures

When tumor size was ˜60 mm³ (day0) the following treatments initiated:

-   -   5. Control (vehicle): Vehicle of Vemurafenib+Trametinib PO—5%        Propylene Glycol, 0.5% Tween-80, 30% PEG 400 in Sterile DDW (5        times/week, qd), 6mice    -   6. Compound D 70 mg/kg in 20% 2-Hydroxypropyl-β-cyclodextrin        (HPbCD), IV (3 times/week, qod), 6 mice    -   7. Vemurafenib 75 mg/kg+Trametinib 1 mg/kg PO (5 times/week,        qd), 20 mice.    -   8. Vemurafenib 75 mg/kg+Trametinib 1 mg/kg PO (5 times/week,        qd)+Compound D 70 mg/kg IV (3 times/week), 7 mice.        Vemurafenib+Trametinib were administered ˜4 hr following        Compound D, when administered on the same days.

All treatments for each of the treatment groups 1-4 were initiatedsimultaneously.

The length (l) and the width (w) of the tumors were measured 4 times aweek and the volumes of the tumors were calculated as follows: v=lw²/2.Graphs represent average tumor volumes with standard errors (standarddeviations/square root of group size). Mice weight and behavior wereexamined at least twice a week.

Results

As shown in FIG. 11, while on treatment with Vemurafenib+Trametinib,tumors aggressively progressed on day6 of treatment (FIG. 11, opensquares), all tumors in the combined treatment with Compound D(Vemurafenib+Trametinib+Compound D) regressed (FIG. 11, open circles).

Vemurafenib was the first mutated-BRaf inhibitor approved by the FDA forthe treatment of melanoma patients harboring mutation in BRaf^(V600).Unfortunately, few months after treatment initiation the patientsdeveloped resistance to Vemurafenib and regressed tumors resurged moreaggressively. Consequently, the combination of mutated-BRaf inhibitorand MEK inhibitor was approved for the treatment of melanoma patientsharboring mutation in BRaf^(V600), but still resistance is acquired. Weshow that two feedback pathways are induced by treatment of cells inculture with Vemurafenib (mutated-BRaf inhibitor) or Trametinib (MEKinhibitor)—the levels of both IRS and phosphorylated STATS increase.Both pathways are central for cell survival, proliferation, metastasisand angiogenesis. Without wishing to be bound by any particular theoryor mechanism of action, Compound D and the compounds of formulae (I-IV)described herein are dual inhibitors of IRS1/2 and Stat3 and, therefore,should antagonize these mechanisms induced by the MAPK pathwayinhibitors (like mutated BRaf inhibitors and MEK inhibitors), synergizewith these inhibitors (with each alone or with the combinations of them)and prevent resistance to these inhibitors.

Example 9: Compound D Synergizes with MEK Inhibitor Trametinib to InduceTumor Regression in Mice Implanted with Tumor from Adenoid CycticCarcinoma Patient Harboring Mutation in BRAF

Experimental system: Patient-derived xenograft (PDX) of Adenoid CycticCarcinoma tumor biopsy subcutaneous implanted into NodScid mice.

I. Animals and Biopsy

-   -   Biopsy: Fresh human primary Adenoid Cyctic Carcinoma tumor        biopsy. Genomic analysis revealed mutated BRaf.    -   Implantation of Adenoid Cyctic Carcinoma RA_148 tumor biopsy        graft into NodScid mice for efficacy study:    -   Implantation of tumor biopsy grafts (P0): Fresh human primary        Adenoid Cyctic Carcinoma tumor biopsy grafts were        sub-cutaneously (SC) implanted into NOD.CB17-Prkdcscid/J        (NodScid mice).    -   Implantation of tumor biopsy grafts (P5) into NodScid female        mice (generated by in-house breeding) for efficacy study, was        performed using the same procedure described above for        implantation of HNSCC.    -   Onset of tumor growth (palpable tumor mass) was detected in all        mice five days following cell injection. After three additional        days, mice developed tumors with an average size of about 65        mm³. The mice were randomly divided into treatment groups        including 5 animals/group. The vehicle-treated group included 4        mice.

II. Treatments and Procedures

Treatment groups included:

-   -   1. Vehicle-control: vehicle of Trametinib (5% Propylene Glycol,        0.5% Tween-80, 30% PEG 400 in Sterile DDW) 200 μl PO (5        times/week, qd).    -   2. Compound D 70 mg/kg in HPbCD, IV (3 times/week, qod).    -   3. Trametinib 1 mg/kg PO (5 times/week, qd).    -   4. Trametinib 1 mg/kg PO (5 times/week, qd)+Compound D 70 mg/kg        IV (3 times/week). Trametinib was administered ˜4 hr following        Compound D, when administered on the same days.

All treatments for each of the treatment groups 1-4 were initiatedsimultaneously on day0, and the study included two phases of treatmentsday0-day13 and day24-day31.

The length (l) and the width (w) of the tumors were measured 2-4 times aweek and the volumes of the tumors were calculated as follows: v=lw²/2.Graphs represent average tumor volumes with standard errors (standarddeviations/square root of group size). Mice weight and behavior wereexamined at least twice a week.

Results

As shown in FIG. 12, Treatment with Trametinib induced tumor regression,but while on treatment after day10 tumors progressed. Combined treatmentof Trametinib and Compound D induced tumor regression and none of thesetumors regrew while on treatment. A second phase treatment on days 24-31induced dramatic tumor regression in all mice treated with Trametinibbut the response was transient and after 4 days of treatment tumorsacquired resistance to the treatment and aggressively progressed whileon treatment. Second phase treatment with the Trametinib+Compound Dcombination induced tumor regression and none of these tumors regrewwhile on treatment.

Conclusion

Although the treatment of the initial tumor with Compound D alone led tomoderate tumor growth inhibition, the combined treatment with Trametiniband Compound D led to dramatic tumor regression and the acquiredresistance to Trametinib was abolished by Compound D. Evidence from theliterature suggests that treatment with Trametinib induces IRSup-regulation leading to resistance by the activation ofIGF1R/IRS-to-AKT survival pathway. Other reports claim Trametinibinduces Stat3 phosphorylation in cancer cells leading to survival andacquired resistance to Trametinib. Without wishing to be bound by anyparticular theory or mechanism of action, Compound D and other compoundsof formulae (I-IV) described herein are dual inhibitors of IRS1/2 andStat3 and, therefore, should antagonize these Trametinib-inducedfeedback mechanisms and prevent resistance.

Example 10: Compound D Re-Sensitizes Gemcitabine-Resistant Tumors toGemcitabine in Mice Implanted with Tumor from a Liver Metastasis of aPancreatic Cancer Patient

Experimental system: Patient-derived xenograft (PDX) of a biopsy ofpancreatic cancer metastasis from the liver, subcutaneous implanted intoNodScid mice.

I. Animals and Biopsy

-   -   Implantation of pancreatic cancer metastasis from the liver        RA_160 tumor biopsy graft (P5) into NodScid mice for efficacy        study: Several weeks following implantation of pancreatic cancer        liver metastasis biopsy graft in mice, tumor cells (P5) were        injected into NodScid mice (generated by in-house breeding),        using the same procedure described above.    -   Onset of tumor growth (palpable tumor mass) was detected ten        days following cell injection. A week later (on da0), 19 mice        with average tumor size of 90 mm³ initiated treatments with        Gemcitabine 25 mg/kg IP twice a week for 35 days. On day11 all        tumors in Gemcitabine-treated mice regressed while the tumors in        all 5 control mice progressed. On day21 the average tumor size        in Gemcitabine-treated group was ˜5 mm³ as compared to 1400 mm³        in the control group.    -   42 days following initiation of treatment with Gemcitabine        resistance developed and regressed tumors progressed. Four days        later 16 mice of the Gemcitabine-treated group with average        tumor volume of ˜110 mm³ were divided to two groups as follows.

II. Treatments

Treatment groups, following resistance to Gemcitabine has been acquired,included:

-   -   1. Gemcitabine 25 mg/kg IP twice a week    -   2. Gemcitabine 25 mg/kg IP+Compound D 70 mg/kg IV, twice a week.

The length (l) and the width (w) of the tumors were measured 2-4 times aweek and the volumes of the tumors were calculated as follows: v=lw²/2.Graphs represent average tumor volumes with standard errors (standarddeviations/square root of group size). Mice weight and behavior wereexamined at least twice a week.

Results

Mice were treated with Gemcitabine for more than a month until regressedtumors acquired resistance to Gemcitabine and progressed. At this pointthe Gemcitabine-treated mice were divided to two groups (FIG. 13): (a)Gemcitabine (□); (b) Gemcitabine+Compound D (◯). Treatments wereinitiated when average tumor size was ˜110 mm³. While all tumors treatedwith Gemcitabine progressed, combined treatment with CompoundD+Gemcitabine led to tumor regression in half of the group, andsignificant tumor growth inhibition in terms of average tumor size ofthe group compared to the Gemcitabine-treated group (FIG. 13A, pvalue=7.35*10⁻⁵).

At the end of the experiment, tumor pieces, similar in size, from threetumors per group were cultured in separate plates to test theirviability and proliferative activity. Nine days later the plates werefixed and stained, showing massive proliferation in theGemcitabine-treated tumors as opposed to a very low to negligibleproliferative activity in the tumors from mice treated withGemcitabine+Compound D (FIG. 13B).

Conclusion

Compound D surprisingly synergized with chemotherapeutic drugGemcitabine to combat resistance developed to Gemcitabine in pancreaticcancer.

Example 11: Compound D Prevents Acquired Resistance to Cetuximab in MiceImplanted with a Tumor from an Adnexal Adeno Carcinoma MetastaticPatient

Experimental system: Patient-derived xenograft (PDX) of human primaryAdnexal adeno metastatic carcinoma biopsy subcutaneous implanted intoNodScid mice.

1. Animals and Biopsy

-   -   Biopsy: fresh human Adnexal adeno carcinoma metastatic (skin)        biopsy (sample ID: RA-162)    -   Implantation of tumor biopsy grafts (P3) into NodScid mice for        efficacy study: When tumors (P2) reached average size of about        1500 mm³, tumor tissue was injected into 50 male NodScid mice,        generated by in-house breeding in the animal facilities of Bar        Ilan University, at the same procedure described in example 1.    -   19 mice whose tumors reached average size of about 90 mm³ were        included in the study. The mice were divided into 4 groups and        the following treatments initiated (day 0):

Control: Vehicle of NT219 (20% HPbCD) 5 mice 50 μl IV twice a weekCompound D 70 mg/kg IV twice a week 6 mice Cetuximab 1 mg/mouse IP twicea week 5 mice Cetuximab (1 mg/mouse IP) + Compound 3 mice D (70 mg/kgIV), twice a week

In the combination group Cetuximab was administered ˜4 hr followingCompound D. The length (l) and the width (w) of the tumors were measured4 times a week and the volumes of the tumors were calculated as follows:v=lw²/2. Graphs represent average tumor volumes with standard errors(standard deviations/square root of group size). Mice weight andbehavior were examined at least once a week.

Results

After 5 days of treatment mice of the control group reached theirendpoint (defined as tumor size above 1.5 cm³) (FIG. 14) and weresacrificed.

Treatment with Cetuximab led to transient tumor growth attenuationfollowed by acquired resistance to Cetuximab, and while on treatmenttumors progressed (day 26 of treatment and onwards).

Combined treatment with Cetuximab+Compound D led to a significant tumorregression, and while the Cetuximab-treated group showed average tumorvolume of >500 mm³—the average tumor volume of the combined treatment(Cetuximab+Compound D) was only 60 mm³ at the end of the experiment(day34).

Example 12

Compound D prevents acquired resistance to the combined treatment ofCetuximab and FOLFIRI (an approved treatment for colon cancer patients)in mice implanted with a tumor from a colon cancer patient. FOLFIRIcontains the following regimen:

-   -   FOL—folinic acid (leucovorin), a vitamin B derivative used as a        “rescue” drug for high doses of the drug methotrexate, but        increases the cytotoxicity of 5-fluorouracil;    -   F—fluorouracil (5-FU), a pyrimidine analog and antimetabolite        which incorporates into the DNA molecule and stops synthesis;        and    -   IRI—irinotecan (Camptosar), a topoisomerase inhibitor, which        prevents DNA from uncoiling and duplicating.

Experimental system: Patient-derived xenograft (PDX) of human primarycolon metastatic carcinoma biopsy subcutaneous implanted into NodScidmice.

1. Animals and Biopsy

-   -   Biopsy: fresh human colon cancer biopsy (sample ID: RA-149)    -   Implantation of tumor biopsy grafts (P4) into NodScid mice for        efficacy study: When tumors (P3) reached average size of about        1500 mm³, tumor tissue was injected into male NodScid mice,        generated by in-house breeding in the animal facilities of Bar        Ilan University, at the same procedure described in example 1.    -   36 mice whose tumors reached average size of about 110 mm³ were        included in the study.

The mice were divided into 7 groups and the following treatmentsinitiated (day 0):

Control: Vehicle of NT219 (20% HPbCD) 5 mice 50 μl IV twice a weekCompound D 70 mg/kg IV twice a week 5 mice Cetuximab 1 mg/mouse IP twicea week 5 mice FOLFIRI IP 5 times a week 5 mice Cetuximab 1 mg/mouse IPtwice a week + 5 mice FOLFIRI IP 5 times a week Cetuximab 1 mg/mouse IPtwice a week + 6 mice FOLFIRI IP 5 times a week + Compound D 70 mg/kg IVtwice a week

In the combination group Cetuximab was administered ˜4 hr followingCompound D.

In colon cancer Cetuximab is not effective as a stand-alone therapy,therefore it is approved for patients in combination with chemotherapylike FOLFIRI. FOLFIRI includes folinic acid (leucoverin), 5FU andIrinotecan.

The length (l) and the width (w) of the tumors were measured 4 times aweek and the volume of the tumors were calculated as follows: v=lw²/2.Graphs represent average tumor volumes with standard errors (standarddeviations/square root of group size). Mice weight and behavior wereexamined at least once a week.

Results

No effect on tumor growth was detected in the group treated by eitherCetuximab, Compound D or FOLFIRI alone. But the combination of Cetuximabwith FOLFIRI with or without Compound D led to a significant regressionof the tumors (FIG. 15).

Following 12 days of treatment the tumors of the Cetuximab+FOLFIRI groupdeveloped resistance to the treatment and progressed, while the tumorsof the Cetuximab+FOLFIRI+Compound D group regressed and haven't acquiredresistance to the treatment (FIG. 15).

Conclusions

The FDA has approved Cetuximab (Erbitux) in combination with FOLFIRIregimen as a first-line treatment for patients with metastaticcolorectal cancer who test negative for the KRAS mutation. Cetuximab asa monotherapy in colorectal cancer is usually not effective. The coloncancer PDX model of RA_149 biopsy is in accordance with the clinicalcondition—Cetuximab as a monotherapy is not effective but withchemotherapy like FOLFIRI it induces dramatic tumor regression. Inaddition, as unfortunately seen with patients, resistance is acquiredand tumors progress. We show that combining this therapy with Compound Dprevents acquired resistance to Cetuximab+FOLFIRI, extending thepositive response.

While certain embodiments of the invention have been illustrated anddescribed, it will be clear that the invention is not limited to theembodiments described herein. Numerous modifications, changes,variations, substitutions and equivalents will be apparent to thoseskilled in the art without departing from the spirit and scope of thepresent invention as described by the claims, which follow.

1-105. (canceled)
 106. A method of treating a tumor that has developedresistance to an Epidermal Growth Factor Receptor (EGFR) inhibitorand/or EGFR antibody, or preventing acquired resistance of a tumor to anEGFR inhibitor and/or EGFR antibody, or preventing or delaying tumorrecurrence following cessation of treatment with an EGFR inhibitorand/or EGFR antibody, the method comprising the step of contacting thetumor with a compound represented by the structure of formula (III)and/or (IV):

wherein R¹, R², R⁵ and R⁶ are independently selected from H, C₁-C₄alkyl, (CH₂CH₂O)_(n)H wherein n is an integer of 1 to 20, acyl and afunctional group that gives rise to hydroxyl upon hydrolysis; R³, R⁷,R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³ and R¹⁴ are independently selected from H,halogen, C₁-C₄ alkyl, C₁-C₄ haloalkyl, CH₂SR^(a) wherein R^(a) isselected from H, C₁-C₄ alkyl, aryl, heterocyclyl, heteroaryl, C₁-C₄alkylaryl, C₁-C₄ alkylheterocyclyl and C₁-C₄ alkylhereroaryl, and OR¹⁶wherein R¹⁶ is H, C₁-C₄ alkyl, (CH₂CH₂O)_(n)H, acyl or a functionalgroup that gives rise to hydroxyl upon hydrolysis; and R⁴ is H or CN;

wherein A is H or CN; Z is S, SO or SO₂; X¹, X², X³, X⁴, X⁵, Y¹ and Y²are each independently selected from H, halogen, alkyl, haloalkyl andOR¹; and Y³ and Y⁴ are each OR¹, wherein each R¹ is independently H,C₁-C₄ alkyl, —(CH₂CH₂O)_(n)H wherein n is an integer of 1 to 20, acyl ora functional group that gives rise to hydroxyl upon hydrolysis,including salts, hydrates, solvates, polymorphs, optical isomers,geometrical isomers, enantiomers, diastereomers, and mixtures thereof;in combination with an EGFR antibody selected from the group consistingof cetuximab, panitumumab, and necitumumab.
 107. The method according toclaim 106, wherein the compound of formula (III) is selected from thegroup consisting of compounds A, B, C, D, E, F, G, H, I and J:

including salts, hydrates, polymorphs, and mixtures thereof.
 108. Themethod according to claim 107, wherein the compound of formula (III) isa compound represented by the structure of formula D:

including salts, hydrates, polymorphs, and mixtures thereof.
 109. Themethod according to claim 106, wherein the compound of formula (IV) isrepresented by the structure of formula (IV-4):

including salts, hydrates, polymorphs, and mixtures thereof.
 110. Themethod according to claim 106, wherein the EGFR antibody is cetuximab.111. The method according to claim 106, wherein the tumor is present ina cancer patient having a tumor with acquired resistance to treatmentwith an EGFR inhibitor and/or EGFR antibody, wherein the treatmentresults in attenuation or regression in the growth of the tumor withacquired resistance to EGFR inhibitor and/or EGFR antibody treatment.112. The method according to claim 106, wherein the tumor is present ina cancer patient who is receiving treatment with an EGFR inhibitorand/or EGFR antibody or is a candidate for receiving such treatment.113. The method according to claim 106, wherein the tumor is present ina patient having a cancer selected from the group consisting of head andneck (H&N) cancer, sarcoma, multiple myeloma, ovarian cancer, breastcancer, kidney cancer, hematopoietic cancer, lung carcinoma, melanoma,glioblastoma, hepatocarcinoma, prostate cancer, thyroid cancer,esophageal carcinoma, stomach cancer, and colon cancer.
 114. The methodaccording to claim 113, wherein the tumor is present in a patient havinghead and neck (H&N) cancer, thyroid cancer, esophageal carcinoma,stomach cancer, or colon cancer.
 115. The method according to claim 113,wherein the tumor is present in a patient having hematopoietic cancer,wherein the hematopoietic cancer is lymphoma or leukemia.
 116. Apharmaceutical combination comprising an Epidermal Growth FactorReceptor (EGFR) antibody selected from the group consisting ofcetuximab, panitumumab, and necitumumab; and a compound represented bythe structure of formula (III) and/or (IV):

wherein R¹, R², R⁵ and R⁶ are independently selected from H, C₁-C₄alkyl, (CH₂CH₂O)_(n)H wherein n is an integer of 1 to 20, acyl and afunctional group that gives rise to hydroxyl upon hydrolysis; R³, R⁷,R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³ and R¹⁴ are independently selected from H,halogen, C₁-C₄ alkyl, C₁-C₄ haloalkyl, CH₂SR^(a) wherein R^(a) isselected from H, C₁-C₄ alkyl, aryl, heterocyclyl, heteroaryl, C₁-C₄alkylaryl, C₁-C₄ alkylheterocyclyl and C₁-C₄ alkylhereroaryl, and OR¹⁶wherein R¹⁶ is H, C₁-C₄ alkyl, (CH₂CH₂O)_(n)H, acyl or a functionalgroup that gives rise to hydroxyl upon hydrolysis; and R⁴ is H or CN;

wherein A is H or CN; Z is S, SO or SO₂; X¹, X², X³, X⁴, X⁵, Y¹ and Y²are each independently selected from H, halogen, alkyl, haloalkyl andOR¹; and Y³ and Y⁴ are each OR¹, wherein each R¹ is independently H,C₁-C₄ alkyl, — (CH₂CH₂O)_(n)H wherein n is an integer of 1 to 20, acylor a functional group that gives rise to hydroxyl upon hydrolysis,including salts, hydrates, solvates, polymorphs, optical isomers,geometrical isomers, enantiomers, diastereomers, and mixtures thereof.117. The pharmaceutical combination according to claim 116, wherein thecompound of formula (III) is selected from the group consisting ofcompounds A, B, C, D, E, F, G, H, I and J:

including salts, hydrates, polymorphs, and mixtures thereof.
 118. Thepharmaceutical combination according to claim 117, wherein the compoundof formula (III) is a compound represented by the structure of formulaD:

including salts, hydrates, polymorphs, and mixtures thereof.
 119. Thepharmaceutical combination according to claim 116, wherein the compoundof formula (IV) is represented by the structure of formula (IV-4):

including salts, hydrates, polymorphs, and mixtures thereof.
 120. Thepharmaceutical combination according to claim 116, wherein the EGFRantibody is cetuximab.
 121. The pharmaceutical combination according toclaim 116, wherein the compound is represented by the structure offormula D:

including salts, hydrates, polymorphs, and mixtures thereof, and whereinthe EGFR antibody is cetuximab.
 122. The pharmaceutical combinationaccording to claim 116, wherein the compound represented by thestructure of formula (III) and/or (IV) is in a form suitable for oraladministration, intravenous administration, topical administration,administration by inhalation, or administration via a suppository; andwherein the EGFR antibody is in a form suitable for intravenousadministration.
 123. The pharmaceutical combination according to claim122, wherein the compound represented by the structure of formula (III)and/or (IV) is formulated in a solution, a suspension, a syrup, anemulsion, a dispersion, a tablet, a pill, a capsule, a pellet, granules,a powder, an ointment, a gel, or a cream; and wherein the EGFR antibodyis formulated in a solution or a suspension.
 124. The pharmaceuticalcombination according to claim 116, wherein the compound represented bythe structure of formula (III) and/or (IV) and the EGFR antibody areadministered in the same pharmaceutical composition.
 125. Thepharmaceutical combination according to claim 116, wherein the compoundrepresented by the structure of formula (III) and/or (IV) and the EGFRantibody are administered in separate pharmaceutical compositions,simultaneously or sequentially, in any order.